Sfpi1/PU.1 mutations in mouse radiation-induced acute myeloid leukaemias affect mRNA and protein abundance and associate with disrupted transcription.

Radiation-induced acute myeloid leukaemias (AMLs) in mice are characterised by deletions and point mutations in the Sfpi1/PU.1 transcription factor. Six AML cell lines were used to examine the impact of three previously described R235 point mutations. AML cells carry myeloid and stem cell markers and the R235 mutations differentially affect mRNA and protein abundance. Expression of Sfpi1/PU.1 target genes was deregulated in a broadly similar fashion irrespective of R235 mutation including Flt3, which is frequently subject to activating mutations in human myeloid leukaemias. While R235 mutations differentially affect protein abundance they resulted in similar disruption of Sfpi1/PU.1 functions.

Tails of the unexpected: palatal medial edge epithelium is no more specialized than other embryonic epithelium.

OBJECTIVE: To determine whether palatal medial edge epithelium (MEE) is specialized in its ability to disappear compared with other embryonic, non-palatal, epithelium. SUBJECTS: Embryonic tissues harvested from CD1 mice. METHODS: Organs were cultured in 2 ml of DMEM/F12 supplemented with 300 microg/ml L-glutamine and 1% penicillin/streptomycin. Organs were cultured under various conditions including opposing other organs and opposing an inert material for a period of 6 days. Tissues were then processed for histological examination. RESULTS: MEE of shelves opposing nothing persisted, whereas MEE of shelves contacting another shelf disappeared. When a tail was placed against a palatal shelf the MEE disappeared, as did the epithelium from the tail, resulting in fusion between the shelf and tail. Furthermore, when palatal shelves were placed against an inert material the MEE disappeared, suggesting pressure alone is a sufficient stimulus to initiate disappearance of the MEE, and that the interaction between the two palatal shelves is not a prerequisite for the disappearance of MEE. Moreover, when two embryonic tails were cultured in close apposition they fused, as did paired limbs. Non-palatal epithelia also disappeared after contact with inert materials. Epithelial disappearance began within 24 h of contact, but there was an age limit. CONCLUSION: These findings suggest that embryonic epithelium from non-specific sites around the body has the ability to disappear with mechanical contact resulting in fusion of tissues. MEE may not be as specialized as once thought.

A new approach for the recovery of precious metals from solution and from leachates derived from electronic scrap.

A new approach is described for the recovery of precious metals (PMs: Au, Pd and Ag) with >99% efficiency from aqueous solution utilising biogas produced during the aerobic growth of Klebsiella pneumoniae. Gold was recovered from electronic scrap leachate ( approximately 95%) by this method, with some selectivity against Cu. The recovered PM solids all contained metal and sulphur as determined by energy dispersive X-ray microanalysis (EDX). X-ray powder diffraction analysis (XRD) showed no crystalline metal sulphur compounds but a crystalline palladium amine was recorded. Silver was recovered as a sulphide (found by EDX), carbonate and oxide (found by XRD). EDX analysis of the Au-precipitate showed mainly gold and sulphur, with some metallic Au(0) detected by XRD. The gold compound was shock-sensitive; upon grinding it detonated to leave a sooty black deposit.

A new approach to the remediation of heavy metal liquid wastes via off-gases produced by Klebsiella pneumoniae M426.

When the off-gas from an aerobic culture of Klebsiella pneumoniae M426 grown in the absence of added heavy metals was passed through a solution of Hg(2+), Cd(2+), Pb(2+), or Cu(2+) a yellow-white (Hg), white (Cd, Pb), or blue (Cu) precipitate was formed. Metal removal from solution was >97% within 2 h at initial concentrations of (as metal): Hg, 8.5; Cd, 12.6; Pb, 7.8; Cu, 9.5 mg/mL. Mercury was removed from solution at pH 2 and in up to 1 M NaCl. Energy dispersive X-ray microanalysis (EDX) of the precipitates showed them to comprise metal, sulfur and carbon in the case of Hg, Cd, and Pb, and, in the case of Cd and Pb, also oxygen. The pH of the solution increased by 1-2 units at an initial pH of 7 and by 4-5 units at an initial pH of 2. In the case of cadmium and lead, the presence of crystalline metal carbonates and hydroxides was confirmed by X-ray powder diffraction (XRD) analysis and additional peaks were seen which could not be assigned to known compounds in the diffraction file database. In the case of copper, hydroxides, and a form of copper sulfate, were found. In the case of mercury the XRD patterns could not be assigned to any known compound, except for HgCl in the solution at the acidic initial pH. The absence of sharp peaks in the pattern for the Hg-precipitate was indicative of poorly crystalline, nanocrystalline or amorphous material. The unknown mercury compound, since it contained non-carbonate carbon, was suggested to be derived from a volatile organothiol in the gases evolved from the culture. Analysis of the culture head gas by GC-MS showed the presence of dimethyldisulfide as a likely precipitant. No sulfur compound was found using XRD analysis in the case of cadmium and lead, although EDX analysis suggested this as a major component and the lack of XRD pattern is evidence for a non-crystalline metal-organothiol. The exact chemistry of the new materials remains to be elucidated but metal precipitation via a biogenic organothiol is a potentially effective approach to the remediation of aggressive metal wastes.

Mercury tolerance of thermophilic Bacillus sp. and Ureibacillus sp.

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 degrees C on Luria agar containing HgCl(2). Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl(2) were 80 microg/ml and 30 microg/ml for these isolates, respectively, compared to 10 microg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 microg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H(2)S was detected in the headspace of cultures in the absence or presence of Hg(2+), and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg(2+) without production of toxic volatile H(2)S.

A paralleling device and ethylene vinyl acetate baffles for use with mandibular distraction osteogenesis: technical note.

A novel method for planning the placement of intra-oral lengthening devices using a paralleling device is described and illustrated with a case report. Simple radiographic measurements and study models are all that is required to construct a simple acrylic splint with guides, which allows accurate positioning of the distractors at surgery. The construction of ethylene vinyl acetate (EVA) baffles to prevent trauma to the labial mucosa from the intra-oral link arms is a technique that enhances patient comfort during distraction of the mandible. The case report demonstrates the application of the surgical planning technique and the use of EVA baffles for a patient with an overjet of 21.5 mm.

Stimulation of microbial sulphate reduction in a constructed wetland: microbiological and geochemical analysis.

Microbial sulphate reduction was stimulated successfully in enclosures installed in a constructed wetland. When sucrose (2.4mM) and NH(4)Cl (600 microM) were added to water in the test enclosures, the indigenous microbial community was able to remove over 90% of the sulphate, present as a contaminant from nearby mining activity at a concentration of 384 mg x l(-1) (4mM), over 50 days. Over 90% of the sucrose was also removed. Sulphate was not reduced in control enclosures containing no added sucrose or NH(4)Cl. Fermentation of sucrose by obligate anaerobes including Clostridium sp. and Bacteriodes sp. preceded sulphate reduction in the test enclosures. Sulphate reduction was biphasic, with maximum rates noted between 2-5 and 23-27 days after the addition of the growth substrates. Relatively unbiased 16S rDNA analysis suggested that nitrogen-fixing bacteria were important constituents of the microbial community in the test enclosures at day 23, suggesting that soluble nitrogen was limiting in the amended test enclosures during the experiment.

Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes.

Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [(125)I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG alphabeta+); and 3) ovariectomized (OVX) hCG alphabeta+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG alphabeta+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG alphabeta+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.

The conservative approach to managing unerupted lower premolars -- two case reports.

General dental practitioners frequently refer patients with unerupted premolars for specialist management. The frequency of unerupted lower second premolars in 15-year-old children has been cited as high as 9.7%. Two cases are discussed involving unilateral unerupted premolars, which initially appear to be in unfavourable positions. The first patient was referred at 16 years of age and presented with an unerupted lower left first premolar positioned along the lower border of the mandible. The second patient presented with an unerupted distally inclined, horizontally positioned second premolar impacting on the roots of the first permanent molar. Both cases were reviewed without any treatment, and both premolars erupted into excellent positions. This raises important questions concerning the possible treatment options for such teeth as well as the timing of any interceptive treatment. In cases where premolars are unerupted or impacted, a multidisciplinary approach is indicated involving orthodontics, paedodontics and oral surgery to establish the optimal treatment plan.

Matrix metalloproteinases have a role in palatogenesis.

Mammalian palatogenesis depends on palatal shelf elevation, medial edge epithelium (MEE) breakdown, and mesenchyme flow. These all require matrix remodeling, which is controlled in part by the family of matrix metalloproteinases (MMPs). We used an organ culture system to examine the effect of a general MMP inhibitor (BB3103) on mouse palatogenesis. Palates cultured in 20 micro M BB3103 contained no active MMP-2, and only one palate fused from a sample size of 15. In this single palate, MMP-3 was present at higher levels than in palates that failed to fuse. MMP-3 is known to be involved in epithelial mesenchymal transformation (EMT), and its persistence may explain why this palate fused. This implies a role for MMPs in normal palatogenesis, and disruption of their activity may result in cleft palate.

Reduction of Cr(VI) and bioaccumulation of chromium by gram positive and gram negative microorganisms not previously exposed to Cr-stress.

Resistance to Cr(VI) is usually associated with its cellular exclusion, precluding enrichment techniques for the isolation of organisms accumulating Cr(VI) via bioreduction to insoluble Cr(III). A technique was developed to screen for potential Cr(VI) reduction in approx. 2000 isolates from a coastal environment, based on the non-specific reduction of selenite and tellurite to Se0 and Te0, and reduction of tetrazolium blue to insoluble blue formazan. The most promising strains were further screened in liquid culture, giving three, which were identified by 16S rRNA sequence analysis as Bacillus pumilus, Exiguobacterium aurantiacum and Pseudomonas synxantha, all of which reduced 100 microM Cr(VI) anaerobically, without growth. The respective removal of Cr(VI) was 90% and 80% by B. pumilus and E. aurantiacum after 48 h and 80% and by P. synxantha after 192 h. With the gram positive strains Cr(VI) promoted loss of flagella and, in the case of B. pumilus, lysis of some cells, but Cr was deposited as an exocellular precipitate which was identified as containing Cr and P using energy dispersive X-ray microanalysis (EDAX). This prompted the testing of Citrobacter sp. N14 (subsequently re-assigned by 16S rRNA sequence analysis and biochemical studies as a strain of Serratia) which bioprecipitates metal cation phosphates via enzymatically-liberated phosphate. This strain reduced Cr(VI) at a rate comparable to that of P. synxantha but Cr(III) was not bioprecipitated where La(III) was removed as LaPO4, even though a similar amount of phosphate was produced in the presence of Cr(III). Since B. pumilus removed most of the Cr(VI), with the formation of cell-bound CrPO4 implicated, this suggests that this strain could have future bioprocess potential.

Mechanisms of mercury bioremediation.

Mercury is one of the most toxic heavy metals, and has significant industrial and agricultural uses. These uses have led to severe localized mercury pollution. Mercury volatilization after its reduction to the metallic form by mercury-resistant bacteria has been reported as a mechanism for mercury bioremediation [Brunke, Deckwer, Frischmuth, Horn, Lunsdorf, Rhode, Rohricht, Timmis and Weppen (1993) FEMS Microbiol. Rev. 11, 145-152; von Canstein, Timmis, Deckwer and Wagner-Dobler (1999) Appl. Environ. Microbiol. 65, 5279-5284]. The reduction/volatilization system requires to be studied further, in order to eliminate the escape of the metallic mercury into the environment. Recently we have demonstrated three different mechanisms for mercury detoxification in one organism, Klebsiella pneumoniae M426, which may increase the capture efficiency of mercury.

Mercury transport and resistance.

Resistance to mercuric ions in bacteria is conferred by mercuric reductase, which reduces Hg(II) to Hg(0) in the cytoplasmic compartment. Specific mercuric ion transport systems exist to take up Hg(II) salts and deliver them to the active site of the reductase. This short review discusses the role of transport proteins in resistance and the mechanism of transfer of Hg(II) between the mercury-resistance proteins.

Mercury resistance (mer) operons in enterobacteria.

Mercury resistance is found in many genera of bacteria. Common amongst enterobacteria are transposons related to Tn21, which is both mercuric ion- and streptomycin-/spectinomycin- and sulphonamide-resistant. Other Tn21-related transposons often have different antibiotic resistances compared with Tn21, but share many non-antibiotic-resistance genes with it. In this article we discuss possible mechanisms for the evolution of Tn21 and related genetic elements.

The effects of a cytokine suppressive anti-inflammatory drug on the output of prostaglandin E(2) and interleukin-1 beta from human fetal membranes.

Fetal membranes are a primary source of prostaglandins and pro-inflammatory cytokines implicated in human parturition, so the inhibition of inflammatory pathways may be of benefit in pregnancies complicated by premature labour. We have therefore investigated the effects of a cytokine-suppressant anti-inflammatory drug (CSAID) on the output of prostaglandin E(2) (PGE(2)) and interleukin (IL)-1 beta from human fetal membranes in vitro. Bacterial endotoxin increased the expression of mRNA for IL-1 beta and type-2 cyclo-oxygenase (COX-2), and there were corresponding increases in the output of IL-1 beta protein and PGE(2). The CSAID decreased IL-1 beta protein, COX-2 expression and PGE(2) output, but not mRNA for IL-1 beta, indicating a post-translational effect on the production of IL-1 beta and a transcriptional affect on COX-2, with an overall reduction in PGE(2). These findings are consistent with the effects of CSAIDs in other systems, and indicate that they are of possible use in premature labour.

Type I collagen synthesis by human osteoblasts in response to placental lactogen and chaperonin 10, a homolog of early-pregnancy factor.

Recent studies have indicated that maternal skeletal metabolism undergoes significant changes during gestation. The agents that are responsible for eliciting these changes in bone turnover during pregnancy have yet to be defined. We therefore sought to investigate whether chaperonin 10 (Cpn10), a homolog of early-pregnancy factor, or human placental lactogen (PL) were capable of influencing the synthesis of type I collagen by human osteoblasts in vitro. Both Cpn10 and PL are major components of the maternal circulation during pregnancy, but how they might contribute to bone metabolism has not been determined. Type I collagen represents the most abundant component of bone tissue, accounting for approximately 90% of the organic compartment. Both Cpn10 and PL were capable of stimulating the synthesis of type I collagen by human osteoblasts in culture. The inclusion of 17 beta-estradiol or prolactin, however, failed to influence the ability of cells to mobilize type I collagen. These novel findings support a role for PL and Cpn10 in the metabolism of bone tissue during pregnancy. Maternal bone collagen metabolism is clearly an important event during pregnancy, and the identification of the factors responsible will aid our understanding of the regulation of skeletal metabolism during gestation.

The role of basic helix-loop-helix genes in vertebrate retinogenesis.

The developing eye is a favorite model for the study of pattern formation and cell fate determination. Retinal neuron development, in particular, is an approachable system to study molecular and cellular aspects of cell determination and differentiation. Basic helix-loop-helix (bHLH) transcription factors are important regulators of retinal neurogenesis. Proneural bHLH genes have highly defined expression in the developing retina that are influenced by pattern formation and cell specification pathways. Each retinal cell class has unique bHLH requirements, implying that these genes regulate neuronal identity and function. Therefore, proneural genes represent a molecular focal point through which epithelial cells are transformed into a precise neural network. In this review, we focus on the bHLH factor Ath5, an important regulator of retinal ganglion cell development, and discuss factors that regulate its expression in the retina and the target genes through which it may confer specific neuronal properties.

Chromate reduction and 16S rRNA identification of bacteria isolated from a Cr(VI)-contaminated site.

A gram-positive, hexavalent chromium [chromate: Cr(VI)]-tolerant bacterium, isolated from tannery waste from Pakistan, was identified as a Microbacterium sp. by 16S rRNA gene sequence homology. The strain (designated as MP30) reduced toxic Cr(VI) only under anaerobic conditions at the expense of acetate as the electron donor. The bacterium was able to grow aerobically in L-broth supplemented with 15 mM CrO4(2-) but then did not reduce Cr(VI). At a concentration of 2.4x10(9) cells/ml, 100 microM sodium chromate was reduced within 30 h; however, the maximum specific reduction rate was obtained at lower initial cell concentrations.

Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a predicted prolipoprotein signal peptidase. Downstream of pbrC, the pbrD gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration. Pb(II)-dependent inducible transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins. This is the first report of a mechanism for specific lead resistance in any bacterial genus.

Math5 is required for retinal ganglion cell and optic nerve formation.

The vertebrate retina contains seven major neuronal and glial cell types in an interconnected network that collects, processes and sends visual signals through the optic nerve to the brain. Retinal neuron differentiation is thought to require both intrinsic and extrinsic factors, yet few intrinsic gene products have been identified that direct this process. Math5 (Atoh7) encodes a basic helix-loop-helix (bHLH) transcription factor that is specifically expressed by mouse retinal progenitors. Math5 is highly homologous to atonal, which is critically required for R8 neuron formation during Drosophila eye development. Like R8 cells in the fly eye, retinal ganglion cells (RGCs) are the first neurons in the vertebrate eye. Here we show that Math5 mutant mice are fully viable, yet lack RGCs and optic nerves. Thus, two evolutionarily diverse eye types require atonal gene family function for the earliest stages of retinal neuron formation. At the same time, the abundance of cone photoreceptors is significantly increased in Math5(-/-) retinae, suggesting a binary change in cell fate from RGCs to cones. A small number of nascent RGCs are detected during embryogenesis, but these fail to develop further, suggesting that committed RGCs may also require Math5 function.