Eukaryotic RNA polymerases are large complexes, 12 subunits of which are structurally or functionally homologous across the three polymerase classes. Each class has a set of specific subunits, likely targets of their cognate transcription factors. We have identified and characterized a human RNA polymerase I (Pol I)-specific subunit, previously identified as ASE-1 (antisense of ERCC1) and as CD3epsilon-associated signal transducer (CAST), and here termed CAST or human Pol I-associated factor of 49 kDa (hPAF49), after mouse orthologue PAF49. We provide evidence for growth-regulated Tyr phosphorylation of CAST/hPAF49, specifically in initiation-competent Pol Ibeta complexes in HeLa cells, at a conserved residue also known to be important for signaling during T-cell activation. CAST/hPAF49 can interact with activator upstream binding factor (UBF) and, weakly, with selectivity factor 1 (SL1) at the rDNA (ribosomal DNA repeat sequence encoding the 18S, 5.8S, and 28S rRNA genes) promoter. CAST/hPAF49-specific antibodies and excess CAST/hPAF49 protein, which have no effect on basal Pol I transcription, inhibit UBF-activated transcription following functional SL1-Pol I-rDNA complex assembly and disrupt the interaction of UBF with CAST/hPAF49, suggesting that interaction of this Pol I-specific subunit with UBF is crucial for activation. Drawing on parallels between mammalian and Saccharomyces cerevisiae Pol I transcription machineries, we advance one model for CAST/hPAF49 function in which the network of interactions of Pol I-specific subunits with UBF facilitates conformational changes of the polymerase, leading to stabilization of the Pol I-template complex and, thereby, activation of transcription.

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.

BACKGROUND

Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described.

RESULTS

A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1) were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents.

CONCLUSIONS

These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations.

About half of Caenorhabditis elegans genes have a 1-2 bp mismatch to the canonical AAUAAA hexamer that signals 3' end formation. One rare variant, AGUAAA, is found at the 3' end of the mai-1 gene, the first gene in an operon also containing gpd-2 and gpd-3. When we expressed this operon under heat shock control, 3' end formation dependent on the AGUAAA was very inefficient, but could be rescued by a single bp change to create a perfect AAUAAA. When AGUAAA was present, most 3' ends formed at a different site, 100 bp farther downstream, right at the gpd-2 trans-splice site. Surprisingly, 3' end formation at this site did not require any observable match to the AAUAAA consensus. It is possible that 3' end formation at this site occurs by a novel mechanism--trans-splicing-dependent cleavage--as deletion of the trans-splice site prevented 3' end formation here. Changing the AGUAAA to AAUAAA also influenced the trans-splicing process: with AGUAAA, most of the gpd-2 product was trans-spliced to SL1, rather than SL2, which is normally used at downstream operon trans-splice sites. However, with AAUAAA, SL2 trans-splicing of gpd-2 was increased. Our results imply that (1) the AAUAAA consensus controls 3' end formation frequency in C. elegans; (2) the AAUAAA is important in determining SL2 trans-splicing events more than 100 bp downstream; and (3) in some circumstances, 3' end formation may occur by a trans-splicing-dependent mechanism.

The complete tropomyosin gene, designated tmy-1, of Caenorhabditis elegans was recovered by genome walking from a fragment that was obtained by exon-expression cloning using specific cloning using specific anti-tropomyosin antiserum as a probe. The genome structure of the tmy-1 gene has been determined by combining the DNA sequences of cDNA clones with those of the genomic fragments. The single-copy gene spans approximately 13 kb and include 14 exons. Comparison of cDNA and genomic sequences demonstrates that three isoforms are encoded by the gene tmy-1. Homology of the 27 C-terminal amino acid residues to those of Drosophila and vertebrates suggest that these may be the body wall, pharyngeal and non-muscle types. Tissue-specific expression of the tmy-1 gene was determined by microinjection of a promoter/lacZ fusion gene and with immunohistochemistry by using affinity-purified tissue-specific anti-tropomyosins. The 5' end promoter common to CeTMI and CeTMII is expressed in the body wall muscles, vulva, anus muscles and male tail muscles. Control sequences of the 5' end promoter are located 660 to 800 bp upstream of the initial methionine codon. The third isoform, CeTMIII, encoding 256 amino acids residues was expressed in the pharyngeal muscles by the promoter in the third intron. The mRNA of CeTMIII was trans-spliced with SL1 and SL2. These results allow is to solve the question of what is common from this worm to vertebrates, and also what are the cross-species complexities and the tissue-specific differences of tropomyosins. The tmy-1 gene is located on the C. elegans genomic YAC grid near the right end of chromosome I, in the region on the lev-11 gene.

We performed in vitro selection of oligoribonucleotides in order to identify high-affinity motifs recognizing RNA hairpins located at the 3' end (SL1) and at the 5' end (domain IV of the internal ribosome entry site) of the hepatitis C virus mRNA. We selected aptamers constituted by an internal loop complementary to the SL1 apical loop, flanked by G-C-rich double-stranded regions, able to form complexes with a K(d) of 70 nM, at 37 degrees C under ionic conditions close to intracellular ones. The complex involves selective apical loop (SL1)-internal loop (aptamer) interactions. Similar structurally organized aptamers were independently identified against domain IV and were shown to also give rise to such complexes. Apical loop-internal loop interaction could constitute a new recognition motif allowing specific intra- or intermolecular RNA-RNA association.

DNA-dependent protein kinase (DNA-PK) is a nuclear enzyme that phosphorylates several transcription factors, but its cellular function has not been elucidated. Here I show that DNA-PK strongly inhibits promoter-directed transcription initiation by Xenopus RNA polymerase I in vitro. The repression is due to protein phosphorylation, since it is relieved by 6-dimethylaminopurine, an inhibitor of protein kinases. DNA-PK inhibits transcription from both linear and circular templates, but the repression is more efficient on linear templates. DNA-PK has no effect on promoter-directed transcription by RNA polymerases II and III. Partial fractionation of the in vitro transcription system shows that a protein fraction containing transcription factor Rib1, the Xenopus equivalent of human SL1, mediates the repression of transcription by DNA-PK. The present data suggest a role for DNA-PK in down-regulating ribosomal gene transcription.

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1-560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.

Efforts were made to generate C57BL/6 cytotoxic effector cells to a syngeneic leukemia (E{male}G2) bearing AKR/Gross virus antigens. As we were unable to induce significant cytotoxic activity by immunization with up to 10(8) irradiated E{male}G2 cells, even when cells from such primed animals were subsequently restimulated with E{male}G2 cells in vitro, C57BL/6 mice were immunized with an aliogeneic, virus-producing AKR leukemic cell line (AKR SL3). Peritoneal exudate cells and, to a lesser degree, spleen cells from these mice showed significant lytic activity toward the immunizing allogeneic tumor but not toward E{male}G2. When spleen cells were harvested from animals {approximately equal to}10 d after injection of AKR SL3 and rechallenged in vitro with either E{male}G2 or AKR.H-2(b) SL1, another tumor that displays AKR/Gross virus antigens, then a vigorous cytotoxic response against E{male}G2 and AKR. H-2(b) SL1 was obtained. Effector cells generated by AKR SL3 priming followed by in vitro stimulation with E{male}G2 or AKR.H-2(b) SL1 lysed only cells of H-2(b) haplotype which were strongly positive for the display of serologically detectable AKR/Gross virus antigens. Thus, AKR SL3 cells were not lysed nor were EL4 cells (H-2(b); but only weakly positive for gp70). Cells not bearing the MuLV antigens tested for, such as P815 mastocytoma cells and spleen cell "blasts" from C57BL/6 and CBA (H-2(k)) mice, were also insusceptible to attack. The cytotoxic effector cells induced bore Thy 1.2 alloantigen and were of the Lyt 1+2+ phenotype. Collectively, these findings are consistent with the conclusion that the cytotoxic T cells raised against E{male}G2 are directed against AKR/Gross virus-associated antigens and are H-2 restricted. It will be of interest to determine the relevance of such effector cells to the known resistance of the C57BL/6 mouse to AKR/Gross virus-induced leukemia.

The saliva of blood sucking insects contains potent pharmacologically active components that assist them in counteracting the host hemostatic and inflammatory systems during blood feeding. In addition, sand fly salivary proteins affect host immunity and have the potential to be a vaccine against Leishmania infection. In the present study, the salivary gland transcripts of Lutzomyia ayacuchensis, a vector of cutaneous leishmaniasis in Ecuadorian and Peruvian Andes, were analyzed by sequencing randomly selected clones of the salivary gland cDNA library of this sand fly. This resulted in the identification of the most abundant transcripts coding for secreted proteins. These proteins were homologous to the salivary molecules present in other sand flies including the RGD-containing peptide, PpSP15/SL1 family protein, yellow-related protein, putative apyrase, antigen 5-related protein, D7 family protein, and 27 kDa salivary protein. Of note, homologues of maxadilan, an active vasodilator abundantly present in saliva of Lutzomyia longipalpis, were not identified. This analysis is the first description of salivary proteins from a sand fly of the subgenus Helcocyrtomyia and from vector of cutaneous leishmaniasis in the New World. The present analysis will provide further insights into the evolution of salivary components in blood sucking arthropods.

Prebiotics and probiotics, either alone or together (synbiotics), can influence the intestinal microbiota and modulate the immune response. We aimed to investigate the effects of prebiotic and synbiotic administration during the early stage of development on the histological structures of central (bursa of Fabricius and thymus) and peripheral (spleen) lymphatic organs in broilers. We used 800 hatching eggs from meat-type hens (Ross 308). Prebiotics and synbiotics were administered in ovo into the air chamber of chicken eggs at d 12 incubation, as follows: prebiotic inulin (Pre1), Bi2tos (Pre2), a synbiotic composed of inulin and Lactococcus lactis subsp. lactis IBB SL1 (Syn1), a synbiotic composed of Bi2tos and L. lactis subsp. cremoris IBB SC1 (Syn2), or physiological saline (control group, C). In ovo delivery of prebiotics and synbiotics had no adverse effect on the development of the immune system in exposed chickens. Administration of Bi2tos with L. lactis subsp. cremoris (Syn2) decreased the cortex/medulla ratio in the thymus and slowed the development of the cortex in bursal follicles on d 21 posthatching, with consequent impacts on the primary lymphatic organs. The above treatment also stimulated germinal centers' formation in the spleens of 21- and 35-day-old chickens, indicating enhanced B-cell proliferation in secondary lymphatic organs. Syn2 also caused an age-dependent increase in the spleen/bursa of Fabricius ratio. In conclusion, the in ovo administration of pre- and synbiotics at d 12 incubation can modulate the central and peripheral lymphatic organ development in broilers. This effect is more pronounced after synbiotic treatment than in prebiotic-treated groups.

The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda.

Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIα in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIα is a component of the initiation-competent RNA polymerase Iβ complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIα functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.

The purpose of this study was to examine how pre- and synbiotic administration in ovo into the air chamber at d 12 of egg incubation influenced the specific immune cell composition and distribution in the ileum, cecal tonsils (CT) and bursa of Fabricius of broilers. The experiment was performed on 800 hatching eggs of the meat-type chickens (Ross 308). Hatching eggs were treated with: prebiotic, consisting of inulin (Pre1) or Bi(2)tos(®) (Pre2); symbiotic, composed of inulin and Lactococcus lactis subsp. lactis IBB SL1 (Syn1) or Bi(2)tos and Lactococcus lactis subsp. cremoris IBB SC1 (Syn2); or physiological saline as a control group. Seven chickens from each treatment group were randomly selected on , 1, 7, and 21 after hatch for tissue collection. Ileum, cecal tonsil and bursa of Fabricius samples were immunohistochemically stained and the proportions of Bu-1(+), CD3(+), CD4(+), CD8α(+) and TCRγδ(+) cells were estimated. It was indicated that the pre- and synbiotics do not adversely affect the development of the GALT of the chicken. The temporary decrease in B-cell number in bursa on d 7 after hatch suggested an increased colonization rate of the peripheral lymphoid organs by these cells after Pre1, Pre2, and Syn2 treatment. In CT at d 7 after hatch more potent colonization of the GALT by T cells was observed in all pre- and synbiotic treated groups and by B cells in both synbiotic-treated groups than those in respective controls. Then, on d 21 in both synbiotic-treated groups, an increase in T-cell number in ileum was also noticed with faster colonization of the CT by B cells. In 21-day-old chickens, both synbiotics exerted stronger stimulatory effect on the GALT colonization by T cells then prebiotics respectively. Similarly, the colonization by B cells was more pronounced in the Syn2 than in the Pre2 group. The data obtained in this study indicated that prebiotics and particularly synbiotics administrated in ovo stimulated GALT development after hatch.

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (psi) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 psi locus to support packaging and viral infectivity in cis. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different psi element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.

Enterobius vermicularis, pinworm, is one of the most common helminths worldwide, infecting nearly a billion people at all socio-economic levels. In prehistoric populations the paleoparasitological findings show a pinworm homogeneous distribution among hunter-gatherers in North America, intensified with the advent of agriculture. This same increase also occurred in the transition from nomad hunter-gatherers to sedentary farmers in South America, although E. vermicularis infection encompasses only the ancient Andean peoples, with no record among the pre-Colombian populations in the South American lowlands. However, the outline of pinworm paleoepidemiology has been supported by microscopic finding of eggs recovered from coprolites. Since molecular techniques are precise and sensitive in detecting pathogen ancient DNA (aDNA), and also could provide insights into the parasite evolutionary history, in this work we have performed a molecular paleoparasitological study of E. vermicularis. aDNA was recovered and pinworm 5S rRNA spacer sequences were determined from pre-Columbian coprolites (4110 BC-AD 900) from four different North and South American archaeological sites. The sequence analysis confirmed E. vermicularis identity and revealed a similarity among ancient and modern sequences. Moreover, polymorphisms were identified at the relative positions 160, 173 and 180, in independent coprolite samples from Tulán, San Pedro de Atacama, Chile (1080-950 BC). We also verified the presence of peculiarities (Splicing leader (SL1) RNA sequence, spliced donor site, the Sm antigen biding site, and RNA secondary structure) which characterise the SL1 RNA gene. The analysis shows that the SL1 RNA gene of contemporary pinworms was present in pre-Columbian E. vermicularis by 6110 years ago. We were successful in detecting E. vermicularis aDNA even in coprolites without direct microscopic evidence of the eggs, improving the diagnosis of helminth infections in the past and further pinworm paleoepidemiological studies.

BACKGROUND

DNA mutations resulting in the McCoy and Swain-Langley polymorphisms have been identified on complement receptor 1 (CR1)-a ligand for rosetting of Plasmodium falciparum-infected RBCs. The molecular identification of the Kna/Knb polymorphism was sought to develop a genotyping method for use in the study of the Knops blood group and malaria.

METHODS

CR1 deletion constructs were used in inhibition studies of anti-Kna. PCR amplification of Exon 29 was followed by DNA sequencing. A PCR-RFLP was developed with NdeI, BsmI, and MfeI for the detection of Kna/Knb, McCa/McCb, and Sl1/Sl2, respectively. Knops phenotypes were determined with standard serologic techniques.

RESULTS

A total of 310 Malian persons were phenotyped for Kna with 200 (64%) Kn(a+) and 110 (36%) Kn(a-). Many of the Kn(a-) exhibited the Knops-null phenotype, that is, Helgeson. The Kna/b DNA polymorphism was identified as a V1561M mutation with allele frequencies of Kna (V1561) 0.9 and Knb (M1561) 0.1.

CONCLUSIONS

The high frequency (18%) of Knb in West African persons suggests that it is not solely a Caucasian trait. Furthermore, because of the high incidence of heterozygosity as well as amorphs, accurate Knops typing of donors of African descent is best accomplished by a combination of molecular and serologic techniques.

Plant-parasitic nematodes are important and cosmopolitan pathogens of crops. Here, we describe the generation and analysis of 1928 expressed sequence tags (ESTs) of a splice-leader 1 (SL1) library from mixed life stages of the root-lesion nematode Pratylenchus penetrans. The ESTs were grouped into 420 clusters and classified by function using the Gene Ontology (GO) hierarchy and the Kyoto KEGG database. Approximately 80% of all translated clusters show homology to Caenorhabditis elegans proteins, and 37% of the C. elegans gene homologs had confirmed phenotypes as assessed by RNA interference tests. Use of an SL1-PCR approach, while ensuring the cloning of the 5' ends of mRNAs, has demonstrated bias toward short transcripts. Putative nematode-specific and Pratylenchus -specific genes were identified, and their implications for nematode control strategies are discussed.

High levels of rRNA synthesis by RNA polymerase I are important for cell growth and proliferation. In vitro studies have indicated that the formation of a stable complex between the HMG box factor [Upstream binding factor (UBF)] and SL1 at the rRNA gene promoter is necessary to direct multiple rounds of Pol I transcription initiation. The recruitment of SL1 to the promoter occurs through protein interactions with UBF and is regulated by phosphorylation of UBF. Here we show that the protein kinase CK2 co-immunoprecipitates with the Pol I complex and is associated with the rRNA gene promoter. Inhibition of CK2 kinase activity reduces Pol I transcription in cultured cells and in vitro. Significantly, CK2 regulates the interaction between UBF and SL1 by counteracting the inhibitory effect of HMG boxes five and six through the phosphorylation of specific serines located at the C-terminus of UBF. Transcription reactions with immobilized templates indicate that phosphorylation of CK2 phosphoacceptor sites in the C-terminal domain of UBF is important for promoting multiple rounds of Pol I transcription. These data demonstrate that CK2 is recruited to the rRNA gene promoter and directly regulates Pol I transcription re-initiation by stabilizing the association between UBF and SL1.

Genomic RNA dimerization is an essential process in the retroviral replication cycle. In vitro, HIV-2 RNA dimerization is mediated at least in part by direct intermolecular interaction at stem-loop 1 (SL1) within the 5'-untranslated leader region (UTR). RNA dimerization is thought to be regulated via alternate presentation and sequestration of dimerization signals by intramolecular base-pairings. One of the proposed regulatory elements is a palindrome sequence (pal) located upstream of SL1. To investigate the role of pal in the regulation of HIV-2 dimerization, we randomized this motif and selected in vitro for dimerization-competent and dimerization-impaired RNAs. Energy minimization folding analysis of these isolated sequences suggests the involvement of pal region in several short-distance intramolecular interactions with other upstream and downstream regions of the UTR. Moreover, the consensus predicted folding patterns indicate the altered presentation of SL1 depending on the interactions of pal with other regions of RNA. The data suggest that pal can act as a positive or negative regulator of SL1-mediated dimerization and that the modulation of base-pairing arrangements that affect RNA dimerization could coordinate multiple signals located within the 5'-UTR.

We report the full coding sequences and the genomic organization of the four genes encoding acetylcholinesterase (AChE) in Caenorhabditis elegans and Caenorhabditis briggsae, in relation to the properties of the encoded enzymes. ace-1 and ace-2, located on chromosome X and I, respectively, encode two AChEs (ACE-1 and ACE-2) that present 35% identity. The C-terminal end of ACE-1 is homologous to the C terminus of T subunits of vertebrate AChEs. ACE-1 oligomerizes into amphiphilic tetramers. ACE-2 has a hydrophobic C terminus of H type. It associates into glycolipid-anchored dimers. In C. elegans and C. briggsae, ace-3 and ace-4 are organized in tandem on chromosome II, with only 356 nt and 369 nt, respectively, between the stop codon of ace-4 (upstream gene) and the ATG of ace-3. ace-3 produces only 5 % of the total AChE activity. It encodes an H subunit that associates into dimers of glycolipid-anchored catalytic subunits, which are highly resistant to the usual AChE inhibitors, and which hydrolyze butyrylthiocholine faster than acetylthiocholine. ACE-4 is closer to ACE-3 (54 % identity) than to ACE-1 or ACE-2. The usual sequence FGESAG surrounding the active serine residue in cholinesterases is changed to FGQSAG in ace-4. ACE-4 was not detected by our current biochemical methods, although the gene is transcribed in vivo. However the level of ace-4 mRNAs is far lower than those of ace-1, ace-2 and ace-3. The ace-2, ace-3 and ace-4 transcripts were found to be trans-spliced by both SL1 and SL2, although these genes are not included in typical operons. The molecular bases of null mutations g72 (ace-2), p1304 and dc2 (ace-3) have been identified.

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5' ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of -10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only -2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer.

BACKGROUND

It has been suggested that free radicals may be implicated in the pathophysiology of acute mountain sickness (AMS) due to their ability to initiate and propagate cell membrane damage (3). Therefore, the present study was designed to: a) investigate the effects of an expedition to high altitude on metabolic indices of free radical-mediated oxidative stress and assess subsequent implications for skeletal/cardiac muscle damage; and b) determine whether these parameters were different in subjects who developed AMS after gradual ascent to 5100 m (base camp, BC) compared with those who remained healthy.

METHODS

There were 19 male volunteers who were examined at rest and after a standardized maximal exercise test at sea level before and after an expedition (SL1/SL2) and during the first morning of arrival at BC. The trek to BC lasted 20+/-5 d.

RESULTS

A mild increase in the Lake Louise AMS score was observed by the end of day 1 at BC (p < 0.05 vs. SL1/SL2). Four subjects developed AMS, which in one subject later progressed to high altitude pulmonary and cerebral edema. The serum concentration of lipid hydroperoxides (LH) increased markedly at rest and after maximal exercise at BC (p < 0.05 vs. SL1/SL2) whereas no changes were observed for plasma malondialdehyde (MDA). Resting serum total phosphocreatine kinase activity (CPK) and myoglobin also increased at BC (p < 0.05 vs. SL1/SL2) whereas cardiac troponin I (cTnI) remained stable. The resting pain threshold decreased and exercise-induced muscle soreness subsequently increased at BC (p < 0.05 vs. SL1/SL2). An association was observed between resting LH and myoglobin at BC (r = 0.45, p < 0.05) and the increase in LH was related to the increase in exercise-induced muscle soreness at BC (r = 0.96, p < 0.05). Further correlations were identified between the AMS score on day 1 at BC and: a) resting/exercise LH (r = 0.63, p < 0.05/r = 0.51, p < 0.05); and b) resting pain threshold at BC (r = -0.58, p < 0.05). Furthermore, subjects with AMS on day 1 at BC were characterized by a greater decrease in the resting pain threshold and greater increase in resting LH, CPK and myoglobin compared with subjects without AMS (p < 0.05). Headache, fatigue, insomnia and general apathy were the most frequently reported symptoms of AMS.

CONCLUSIONS

Localized free radical-mediated vascular damage of the blood-brain barrier in addition to systemic tissue damage causing overt skeletal muscle soreness may have contributed to the pathophysiology of AMS, the latter through its indirect effects on other non-specific constitutional symptoms such as fatigue and insomnia causing a deterioration in physical performance.

Prebiotics and probiotics applied alone or together (synbiotics) can influence the intestinal microbiota and modulate the immune response. We analyzed the impact of in ovo administration of synbiotics on immune system development in Ross (broiler) and Green-legged Partridgelike (GP, dual-purpose fowl) chickens. For in ovo delivery on the 12th day of the eggs incubation, two strains of lactic acid bacteria (LAB) were used, i.e. Lactococcus lactis subsp. lactis IBB SL1 (S1) and Lactococcus lactis subsp. cremoris IBB SC1 (S2), combined with raffinose family oligosaccharides (RFO) prebiotic. Other treatments included in ovo delivery of commercial synbiotic (S3), RFO prebiotics alone (P) and physiological saline (C). Immune system development was analyzed by relative weight (indices) and histology of the lymphatic organs (bursa of Fabricius, thymus and spleen) at two time points (3rd and 6th week of life). The results indicate that the development of the lymphatic organs was significantly affected by in ovo treatment. The bursa and bursa to spleen index was higher in P and S2 groups of broilers (P < 0.05) when compared to S3. In GP at the 3rd week of age, the spleen index was significantly higher in S2 (P < 0.05). The histological image of the thymus displayed an increase of thymocytes in the cortex in all synbiotic-treated groups (S1, S2, S3). In ovo delivery of synbiotics is an efficient mode of immune system stimulation in chickens but its efficiency depends on chicken genotype.