Protein SUMOylation is Involved in Cell‐cycle Progression and Cell Morphology in Giardia lamblia

The unicellular protozoa Giardia lamblia is a food‐ and waterborne parasite that causes giardiasis. This illness is manifested as acute and self‐limited diarrhea and can evolve to long‐term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin‐like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked‐down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti‐SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α‐tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites.     

Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene

Genes essential for the production of a linear, bacterial (1-->3)-beta- glucan, curdlan, have been cloned for the first time from Agrobacterium sp. ATCC31749. The genes occurred in two, nonoverlapping, genomic fragments that complemented different sets of curdlan( crd )-deficient transposon-insertion mutations. These were detected as colonies that failed to stain with aniline blue, a (1-->3)-beta-glucan specific dye. One fragment carried a biosynthetic gene cluster (locus I) containing the putative curdlan synthase gene, crdS, and at least two other crd genes. The second fragment may contain only a single crd gene (locus II). Determination of the DNA sequence adjacent to several locus I mutations revealed homology to known sequences only in the cases of crdS mutations. Complete sequencing of the 1623 bp crdS gene revealed highest similarities between the predicted CrdS protein (540 amino acids) and glycosyl transferases with repetitive action patterns. These include bacterial cellulose synthases (and their homologs), which form (1-->4)-beta-glucans. No similarity was detected with putative (1-->3)- beta-glucan synthases from yeasts and filamentous fungi. Whatever the determinants of the linkage specificity of these beta-glucan synthases might be, these results raise the possibility that (1-->3)-beta-glucans and (1-->4)-beta-glucans are formed by related catalytic polypeptides.      

Evidence for effect of random genetic drift on G+C content after lateral transfer of fucose pathway genes to Escherichia coli K-12

The cps cluster of Escherichia coli K-12 comprises genes involved in synthesis of capsular polysaccharide colanic acid. Part of the E. coli K-12 cps region has been cloned and sequenced and compared to its Salmonella enterica LT2 counterpart. The cps genes from the two organisms are homologous; in the case of the LT2 genes, with G+C content of 0.61 and codons characteristic of high G+C species, it seems clear that they have been acquired relatively recently by lateral transfer from a high G+C species. The K-12 form of these cps genes is closely related to those of LT2 so must derive from the same high G+C species, but it appears to have transferred much earlier such that random genetic drift has brought P3 (the corrected G+C content of codon base 3) down from 0.77 to 0.64, more than halfway to the E. coli average of 0.57. We estimate, using an equation developed by Sueoka, that the lateral transfer to E. coli took place approximately 45 million years ago. This is the first report we are aware of demonstrating the expected adjustment of P3 after lateral transfer between species with different G+C content DNA.      

Biochemical differentiation in bile duct cestodes and their marsupial hosts

Isozyme electrophoresis was used to assess possible cospeciation of parasites (cestodes of the Progamotaenia festiva complex) and their hosts (Australian diprotodont marsupials) and to compare the extent of interspecific genetic diversity of the parasites and their hosts. On the basis of morphology, there are three species in the complex, although electrophoresis revealed 14 distinct genetic types, most of which were host specific, although there were three cases of apparent host switching. The evolutionary relationships among the parasites were only partially concordant with those among the hosts. Moreover, the extent of electrophoretic diversity among the parasites was much higher than that among hosts.      

A CDPK isoform participates in the regulation of nodule number in Medicago truncatula

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.     

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

Background

To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).

Methods

Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.

Results

We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.

Conclusion

Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.     

Assignment by deletion mapping of the steroid sulfatase X-linked ichthyosis locus to Xp223

Summary A male child and his mother who are nullisomic and monosomic, respectively, for the distal portion of Xp because of an unbalanced X-Y translocation were tested for steroid sulfatase activity after clinical examination had yielded evidence for ichthyosis in the boy. Deficiency of steroid sulfatase was found in the male patient, while in his mother enzyme levels were in the heterozygous range. These results, based on cytogenetic evidence obtained with an elongation technique, indicate that the STS locus is at Xp223.     

Fishers' knowledge and seahorse conservation in Brazil

From a conservationist perspective, seahorses are threatened fishes. Concomitantly, from a socioeconomic perspective, they represent a source of income to many fishing communities in developing countries. An integration between these two views requires, among other things, the recognition that seahorse fishers have knowledge and abilities that can assist the implementation of conservation strategies and of management plans for seahorses and their habitats. This paper documents the knowledge held by Brazilian fishers on the biology and ecology of the longsnout seahorse Hippocampus reidi. Its aims were to explore collaborative approaches to seahorse conservation and management in Brazil; to assess fishers' perception of seahorse biology and ecology, in the context evaluating potential management options; to increase fishers' involvement with seahorse conservation in Brazil. Data were obtained through questionnaires and interviews made during field surveys conducted in fishing villages located in the States of Piauí, Ceará, Paraíba, Maranhão, Pernambuco and Pará. We consider the following aspects as positive for the conservation of seahorses and their habitats in Brazil: fishers were willing to dialogue with researchers; although captures and/or trade of brooding seahorses occurred, most interviewees recognized the importance of reproduction to the maintenance of seahorses in the wild (and therefore of their source of income), and expressed concern over population declines; fishers associated the presence of a ventral pouch with reproduction in seahorses (regardless of them knowing which sex bears the pouch), and this may facilitate the construction of collaborative management options designed to eliminate captures of brooding specimens; fishers recognized microhabitats of importance to the maintenance of seahorse wild populations; fishers who kept seahorses in captivity tended to recognize the condtions as poor, and as being a cause of seahorse mortality.