Systemic injection of AAV9 carrying a periostin promoter targets gene expression to a myofibroblast-like lineage in mouse hearts after reperfused myocardial infarction.

Adeno-associated virus (AAV) has been used to direct gene transfer to a variety of tissues, including heart, liver, skeletal muscle, brain, kidney and lung, but it has not previously been shown to effectively target fibroblasts in vivo, including cardiac fibroblasts. We constructed expression cassettes using a modified periostin promoter to drive gene expression in a cardiac myofibroblast-like lineage, with only occasional spillover into cardiomyocyte-like cells. We compared AAV serotypes 6 and 9 and found robust gene expression when the vectors were delivered by systemic injection after myocardial infarction (MI), with little expression in healthy, non-infarcted mice. AAV9 provided expression in a greater number of cells than AAV6, with reporter gene expression visible in the cardiac infarct and border zones from 5 to 62 days post MI, as assessed by luciferase and Cre-activated green fluorescent protein expression. Although common myofibroblast markers were expressed in low abundance, most of the targeted cells expressed myosin IIb, an embryonic form of smooth muscle myosin heavy chain that has previously been associated with myofibroblasts after reperfused MI. This study is the first to demonstrate AAV-mediated expression in a potentially novel myofibroblast-like lineage in mouse hearts post MI and may open new avenues of gene therapy to treat patients surviving MI.

Mutation of the ST7 tumor suppressor gene on 7q31.1 is rare in breast, ovarian and colorectal cancers.

The gene ST7 has recently been implicated as the broad-range tumor suppressor on human chromosome 7q31.1. We did not detect somatic mutations in ST7 in any of 149 primary ovarian, breast or colon carcinomas. These data suggest that epigenetic downregulation or haploinsufficiency, rather than somatic genetic alterations, may be the primary mechanism of abrogation of ST7 function in these tumor types.

Pathogens: bacterial needles ruled to length and specificity.

The mechanisms behind length regulation of prokaryotic surface structures has long eluded microbiologists. The recent identification of a protein that functions as a 'molecular ruler' to determine the physical length of a bacterial extracellular needle advances our understanding of surface structure biogenesis.

The archaeal flagellum: a different kind of prokaryotic motility structure.

The archaeal flagellum is a unique motility apparatus distinct in composition and likely in assembly from the bacterial flagellum. Gene families comprised of multiple flagellin genes co-transcribed with a number of conserved, archaeal-specific accessory genes have been identified in several archaea. However, no homologues of any bacterial genes involved in flagella structure have yet been identified in any archaeon, including those archaea in which the complete genome sequence has been published. Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins. Also unlike bacterial flagellins but similar to type IV pilins, archaeal flagellins are initially synthesized with a short leader peptide that is cleaved by a membrane-located peptidase. With recent advances in genetic transfer systems in archaea, knockouts have been reported in several genes involved in flagellation in different archaea. In addition, techniques to isolate flagella with attached hook and anchoring structures have been developed. Analysis of these preparations is under way to identify minor structural components of archaeal flagella. This and the continued isolation and characterization of flagella mutants should lead to significant advances in our knowledge of the composition and assembly of archaeal flagella.

Asymptomatic kidney stones in long-term survivors of childhood acute lymphoblastic leukemia.

We hypothesized an association between renal calculi and bone mineral density (BMD) deficits, shown in adults, exists in survivors of childhood acute lymphoblastic leukemia (ALL). Thus, we analyzed the associations between quantitative computed tomography (QCT)-determined renal calcifications and clinical parameters (gender, race, age at diagnosis and age at the time of QCT), BMD, treatment exposures and Tanner stage. We investigated the associations between stone formation and nutritional intake, serum and urinary calcium and creatinine levels, and urinary calcium/creatinine ratio. Exact chi(2)-test was used to compare categorical patient characteristics, and the Wilcoxon-Mann-Whitney test to compare continuous measurements. Of 424 participants, 218 (51.4%) were males; 371 (87.5%) were nonblack. Most (n=270; 63.7%) were >or=3.5 years at ALL diagnosis. Mean (s.d.) and median (range) BMD Z-scores of the entire cohort were -0.4 (1.2) and -0.5 (-3.9 to 5.1), respectively. Nineteen participants (10 males; 10 Caucasians) had kidney stones (observed prevalence of 4.5%; 19/424) with a significant negative association between stone formation and body habitus (body mass index, P=0.003). Stone formation was associated with treatment protocol (P=0.009) and treatment group (0.007). Thus, kidney stones in childhood ALL survivors could herald the future deterioration of renal function and development of hypertension. Long-term follow-up imaging may be warranted in these patients to monitor for progressive morbidity.

Characterization of flagellum gene families of methanogenic archaea and localization of novel flagellum accessory proteins.

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.

Developing world. Patients' choice of anaesthetic techniques in a developing country.

A study of the choice between general and local anaesthetic techniques among 200 post-operative Nigerian patients was conducted, using a questionnaire. The patients were interviewed between two and seven days after various types of surgery. Most of the patients (59.5%) preferred general anaesthesia. The commonest reason was fear with psychological upset if they are awake during surgery. This appears to be a basic human feeling which does not appear to be related to sophistication or level of development. Adequate pre-operative communication between doctors and patients should minimise this apprehension. 18% preferred local anaesthetic techniques while 21.5% would not mind either.

Insertional inactivation of the flaH gene in the archaeon Methanococcus voltae results in non-flagellated cells.

The marine methanogen Methanococcus voltae possesses two transcriptional units that encode a total of four flagellins. Immediately downstream of the flagellin genes are a number of ORFs, some of which are cotranscribed with the flagellin genes. These putative genes have been named flaCDEFGHIJ, although no biochemical data has implicated them in flagellar morphogenesis. None of the flaC-J genes has homology to any bacterial gene, with the exception of flaI, which shows homology to pilT, a gene that encodes a nucleotide binding protein of the type IV pilus family. In this study, insertional mutations in flaH of M. voltae were identified. The mutants were non-motile and non-flagellated as determined by electron microscopy. Southern hybridization experiments confirmed the insertion of a mutagenic vector into flaH and indicated that two, tandem, copies of the vector were present. It is believed that insertion of the vector into flaH should disrupt the transcription of flaIJ due to polar effects. The flaH mutant displayed the same pattern of multiple mRNA transcripts, all originating upstream of flaB1, as the wild-type cells. Northern hybridization experiments failed to detect a flaHIJ transcript in either wild-type or mutant cells. Immunoblotting experiments indicated, however, that the mutants produced similar amounts of flagellin, FlaD and FlaE to wild-type cells. Flagellin localization experiments suggest that the flaH mutant is deficient in flagellin secretion and/or assembly. The mutant also displayed similar preflagellin peptidase activity to the wild-type cells, indicating that none of the genes flaHIJ is likely to be the gene that encodes this enzyme, which is required for cleaving the leader peptide from the preflagellins prior to their incorporation into the flagellar filament. This is the first data indicating that the flaHIJ gene cluster is essential for flagellation in methanogens.

Identification of amino acids in the leader peptide of Methanococcus voltae preflagellin that are important in posttranslational processing.

Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the -1 position is always held by a glycine while the -2 and -3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the -1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the -2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the -3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.

Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii.

The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1. is a member of the Siphoviridae family with a B1 morphology. It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 x 10(7) daltons. BCJA1 was stable over the pH range of 6-11. A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40. The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36500 and 28000, respectively. The genome size was estimated to be between 32.1 and 34.8 kb. The percent G + C content of purified bacteriophage DNA was 45.6. The wildtype bacteriophage is temperate but a clear plaque mutant was isolated.

Isolation and characterization of flagella and flagellin proteins from the Thermoacidophilic archaea Thermoplasma volcanium and Sulfolobus shibatae.

Isolated flagellar filaments of Sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have M(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. The flagellar filaments of Thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an M(r) of 41,000 and which also stained positively for glycoprotein. N-terminal amino acid sequencing indicated that 18 of the N-terminal 20 amino acid positions of the 41-kDa flagellin of T. volcanium were identical to those of the Methanococcus voltae 31-kDa flagellin. Both flagellins of S. shibatae had identical amino acid sequences for at least 23 of the N-terminal positions. This sequence was least similar to any of the available archaeal flagellin sequences, consistent with the phylogenetic distance of S. shibatae from the other archaea studied.

Refinement of an ovarian cancer tumour suppressor gene locus on chromosome arm 22q and mutation analysis of CYP2D6, SREBP2 and NAGA.

Loss of heterozygosity on chromosome 22q was detected in 53% of 123 ovarian carcinomas, suggesting the presence of at least one tumour suppressor gene. We have refined the location of one possible tumour suppressor gene to the region between the microsatellite markers D22S299 and CYP2D. Located within this region are the genes SREBP2 (sterol regulatory element binding protein 2) and NAGA (N-acetyl-alpha-D-galactosaminidase). Investigation of the coding exons of these genes by single stranded conformational polymorphism/heteroduplex analysis failed to identify any somatic genetic alterations in 57 ovarian tumours which exhibited LOH on 22q13. The CYP2D gene locus straddles the distal boundary of the candidate region. Germline variants of the active CYP2D6 gene with differing abilities to metabolise specific substrates have been implicated in the development of various cancers. Comparison of the frequency of the two common germline mutations among 258 ovarian tumours and 231 non-cancer controls did not reveal any significant differences between the two groups. This suggests that the known polymorphic variants of CYP2D6 are not involved in ovarian cancer predisposition. We also conclude that neither NAGA nor SREBP2 are likely to be mutated in ovarian carcinomas.

Slow induction of RecA by DNA damage in Mycobacterium tuberculosis.

In mycobacteria, as in most bacterial species, the expression of RecA is induced by DNA damage. However, the authors show here that the kinetics of recA induction in Mycobacterium smegmatis and in Mycobacterium tuberculosis are quite different: whilst maximum expression in M. smegmatis occurred 3-6 h after addition of a DNA-damaging agent, incubation for 18-36 h was required to reach peak levels in M. tuberculosis. This is despite the fact that the M. tuberculosis promoter can be activated more rapidly when transferred to M. smegmatis. In addition, it is demonstrated that in both species the DNA is sufficiently damaged to give maximum induction within the first hour of incubation with mitomycin C. The difference in the induction kinetics of recA between the two species was mirrored by a difference in the levels of DNA-binding-competent LexA following DNA damage. A decrease in the ability of LexA to bind to the SOS box was readily detected by 2 h in M. smegmatis, whilst a decrease was not apparent until 18-24 h in M. tuberculosis and then only a very small decrease was observed.