Gene transfer and expression of a non-viral polycation-based vector in CD4(+) cells

CD4-selective targeting of an antibody-polycation-DNA complex was investigated The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine(268) (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLL-DNA complexes inhibited the binding of I-125-B-F5 to CD4(+) Jurkat cells, while complexes synthesised either without B-F5 or using a non-specific mouse IgG1 antibody had little or no effect Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was defected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3 complex in the CD4(-) K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a nonspecific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4(+) cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4(+) T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4(+) cells in vivo.

Regrow or repair: Potential regenerative therapies for the kidney

Regenerative medicine is being heralded in a similar way as gene therapy was some 15 yr ago. it is an area of intense excitement and potential, as well as myth and disinformation. However, with the increasing rate of end-stage renal failure and limited alternatives for its treatment, we must begin to investigate seriously potential regenerative approaches for the kidney. This review defines which regenerative options there might be for renal disease, summarizes the progress that has been made to date, and investigates some of the unique obstacles to such treatments that the kidney presents. The options discussed include in situ organ repair via bone marrow recruitment or dedifferentiation; ex vivo stem cell therapies, including both autologous and nonautologous options; and bioengineering approaches for the creation of a replacement organ.

Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia

Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb(-/-)). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb(-/-) mice are healthy and fertile. Despite appearing overtly normal, Vegfb(-/-) hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis. The full text of this article is available at http://www.circresaha.org.

The use of complementary and alternative therapies by patients with cancer

The aim of this research was to assess the prevalence and predictors of complementary and alternative therapy (CAT) use among cancer patients in Australia. A total of 1492 cancer patients attending nine major public cancer treatment centers in New South Wales, Australia, were asked to complete the Supportive Care Needs Survey. Of the 1354 consenting patients, 888 (65%) returned a completed survey. This article reports the secondary analyses of the survey data, specifically focusing on CAT use. For all cancers, 17.1% of patients were using at least one CAT. The two main demographic characteristics of CAT users were gender and age, where females were more likely to use CAT than males and that CAT use declined as age increased. Time since diagnosis was identified as the only significant clinical predictor of CAT use, where CAT use increased with time until 5 years since diagnosis. Our research shows that herbal treatments and naturopathy are the most popular CAT used by cancer patients (constituting over 30% of all CAT use recorded). The use of CAT among cancer patients is a significant issue in cancer care, especially considering the potential interactions between CAT and conventional medicines. Given that many cancer patients may not be aware of potential risks associated with these interactions it is important that oncologists and others involved in cancer patient care are informed about CAT and its use amongst their patients.

Molecular phylogeny of Wolbachia endosymbionts in southeast asian mosquitoes (Diptera: Culicidae) based on wsp gene sequences

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts. Insect-associated Wolbachia form a monophyletic clade in the α-Proteobacteria and recently have been separated into two supergroups (A and B) and 19 groups. Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia. Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences. Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup. Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups.

wsp Gene sequences from the Wolbachia of filarial nematodes

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences.

Gene organization of the dnaA region of Wolbachia

The dnaA region of Wolbachia, an intracellular bacterial parasite of insects, is unique. A glnA cognate was found upstream of the dnaA gene, while neither of the two open reading frames detected downstream of dnaA has any homologue in the database. This unusual gene arrangement may reflect requirements associated with the unique ecological niche this agent occupies.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Selection bias in gene extraction on the basis of microarray gene-expression data

In the context of cancer diagnosis and treatment, we consider the problem of constructing an accurate prediction rule on the basis of a relatively small number of tumor tissue samples of known type containing the expression data on very many (possibly thousands) genes. Recently, results have been presented in the literature suggesting that it is possible to construct a prediction rule from only a few genes such that it has a negligible prediction error rate. However, in these results the test error or the leave-one-out cross-validated error is calculated without allowance for the selection bias. There is no allowance because the rule is either tested on tissue samples that were used in the first instance to select the genes being used in the rule or because the cross-validation of the rule is not external to the selection process; that is, gene selection is not performed in training the rule at each stage of the cross-validation process. We describe how in practice the selection bias can be assessed and corrected for by either performing a cross-validation or applying the bootstrap external to the selection process. We recommend using 10-fold rather than leave-one-out cross-validation, and concerning the bootstrap, we suggest using the so-called. 632+ bootstrap error estimate designed to handle overfitted prediction rules. Using two published data sets, we demonstrate that when correction is made for the selection bias, the cross-validated error is no longer zero for a subset of only a few genes.

Evidence for persistent dysfunction of wild-type aldosterone synthase gene in glucocorticoid-treated familial hyperaldosteronism type I

Background In familial hyperaldosteronism type I (FH-I), glucocorticoid treatment suppresses adrenocorticotrophic hormone-regulated hybrid gene expression and corrects hyperaldosteronism. Objective To determine whether the wild-type aldosterone synthase genes, thereby released from chronic suppression, are capable of functioning normally. Methods We compared mid-morning levels of plasma potassium, plasma aldosterone, plasma renin activity (PRA) and aldosterone : PRA ratios, measured with patients in an upright position, and responsiveness of aldosterone levels to infusion of angiotensin II (AII), for 11 patients with FH-I before and during long-term (0.8-14.3 years) treatment with 0.25-0.75 mg/day dexamethasone or 2.5-10 mg/day prednisolone. Results During glucocorticoid treatment, hypertension was corrected in all. Potassium levels, which had been low (< 3.5 mmol/l) in two patients before treatment, were normal in all during treatment (mean 4.0 +/- 0.1 mmol/l, range 3.5-4.6). Aldosterone levels during treatment [13.2 +/- 2.1 ng/100 ml (mean +/- SEM)] were lower than those before treatment (20.1 +/- 2.5 ng/100 ml, P < 0.05). PRA levels, which had been suppressed before treatment (0.5 +/- 0.2 ng/ml per h), were unsuppressed during treatment (5.1 +/- 1.5 ng/ml per h, P < 0.01) and elevated (> 4 ng/ml per h) in six patients. Aldosterone : PRA ratios, which had been elevated (> 30) before treatment (101.1 +/- 25.9), were much lower during treatment (4.1 +/- 1.0, P < 0.005) and below normal (< 5) in eight patients. Surprisingly, aldosterone level, which had not been responsive (< 50% rise) to infusion of AII for all 11 patients before treatment, remained unresponsive for 10 during treatment. Conclusions Apparently regardless of duration of glucocorticoid treatment in FH-I, aldosterone level remains poorly responsive to AII, with a higher than normal PRA and a low aldosterone : PRA ratio. This is consistent with there being a persistent defect in functioning of wild-type aldosterone synthase gene. (C) Rapid Science Publishers ISSN 0263-6352.

Identification of two novel mutations in the hydroxymethylbilane synthase gene in three patients from two unrelated families with acute intermittent porphyria

We have screened the hydroxymethylbilane synthase cDNAs of 3 patients from 2 families suffering from acute intermittent porphyria (AIP) from Scotland and South Africa using heteroduplex and chemical cleavage of mismatch analyses, Direct sequencing was used to characterise the mutations, The two novel mutations identified were a missense mutation at nucleotide position 64 in exon 3 (R22C) and a single base-pair deletion in exon 15, These mutations are predicted to affect the normal function of the enzyme and, therefore, are expected to be the primary cause of disease in these patients.

Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus

The nifH gene sequence of the nitrogen-fixing bacterium Acetobacter diazotrophicus was determined with the use of the polymerase chain reaction and universal degenerate oligonucleotide primers. The gene shows highest pair-wise similarity to the nifH gene of Azospirillum brasilense. The phylogenetic relationships of the nifH gene sequences were compared with those inferred from 16S rRNA gene sequences. Knowledge of the sequence of the nifH gene contributes to the growing database of nifH gene sequences, and will allow the detection of Acet. diazotrophicus from environmental samples with nifH gene-based primers.

Concordance between dispersal and mitochondrial gene flow: isolation by distance in a tropical teleost, Lates calcarifer (Australian barramundi)

Patterns of population subdivision and the relationship between gene flow and geographical distance in the tropical estuarine fish Lares calcarifer (Centropomidae) were investigated using mtDNA control region sequences. Sixty-three putative haplotypes were resolved from a total of 270 individuals from nine localities within three geographical regions spanning the north Australian coastline. Despite a continuous estuarine distribution throughout the sampled range, no haplotypes were shared among regions. However, within regions, common haplotypes were often shared among localities. Both sequence-based (average Phi(ST)=0.328) and haplotype-based (average Phi(ST)=0.182) population subdivision analyses indicated strong geographical structuring. Depending on the method of calculation, geographical distance explained either 79 per cent (sequence-based) or 23 per cent (haplotype-based) of the variation in mitochondrial gene flow. Such relationships suggest that genetic differentiation of L. calcarifer has been generated via isolation-by-distance, possibly in a stepping-stone fashion. This pattern of genetic structure is concordant with expectations based on the life history of L. calcarifer and direct studies of its dispersal patterns. Mitochondrial DNA variation, although generally in agreement with patterns of allozyme variation, detected population subdivision at smaller spatial scales. Our analysis of mtDNA variation in L. calcarifer confirms that population genetic models can detect population structure of not only evolutionary significance but also of demographic significance. Further, it demonstrates the power of inferring such structure from hypervariable markers, which correspond to small effective population sizes.