Anti-apoptotic gene Bcl2 is required for stapes development and hearing.

In this paper we describe novel and specific roles for the apoptotic regulators Bcl2 and Bim in hearing and stapes development. Bcl2 is anti-apoptotic while Bim is pro-apoptotic. Characterization of the auditory systems of mice deficient for these molecules revealed that Bcl2⁻/⁻ mice suffered severe hearing loss. This was conductive in nature and did not affect sensory cells of the inner ear, with cochlear hair cells and neurons present and functional. Bcl2⁻/⁻ mice were found to have a malformed, often monocrural, porous stapes (the small stirrup-shaped bone of the middle ear), but a normally shaped malleus and incus. The deformed stapes was discontinuous with the incus and sometimes fused to the temporal bones. The defect was completely rescued in Bcl2⁻/⁻Bim⁻/⁻ mice and partially rescued in Bcl2⁻/⁻Bim⁺/⁻ mice, which displayed high-frequency hearing loss and thickening of the stapes anterior crus. The Bcl2⁻/⁻ defect arose in utero before or during the cartilage stage of stapes development. These results implicate Bcl2 and Bim in regulating survival of second pharyngeal arch or neural crest cells that give rise to the stapes during embryonic development.

Screening of tetrachlorodibenzo- p-dioxin-degrading fungi capable of producing extracellular peroxidases under various conditions.

Forty-six pulp-bleaching fungi were screened for production of key enzymes for conversion of polychlorinated dibenzo-p-dioxins--lignin peroxidase (LiP), manganese peroxidase (MnP), and manganese-independent peroxidase (MiP)--under various conditions that would allow their utilization in the environment. Of 38 MnP-producing strains with MiP activity, 22 produced LiP. Three of the new isolates, Bjerkandera sp. strains MS191, MS325, and MS1167, were the best producers of the three different peroxidases, and had reasonable growth rates. The most promising Bjerkandera sp. strain, MS325, exhibited significant levels of LiP and MnP activities under various conditions, e.g., nutrient nitrogen-sufficient or -limited conditions, conditions with or without Mn(II), and changes in temperature (15-37 degrees C). Furthermore, the ability of this strain to degrade 1,3,6,8-tetrachlorodibenzo- p-dioxin was confirmed. The results presented here indicate that utilization of Bjerkandera sp. strain MS325 on a practical scale in the environment has several advantages over many white rot fungi, which produce extracellular peroxidases only under specific conditions such as nutrient limitation.

Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins.

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.

Prenatal diagnosis of 22q11.2 deletion when ultrasound examination reveals a heart defect.

PURPOSE: The incidence of 22q11.2 deletion syndrome is approximately 1 in 5,000 births, and accounts for 5-30% of all heart defects, making it one of the more common genetic conditions in the population. METHODS: We employed fluorescence in situ hybridization (FISH) to study the incidence of 22q11.2 deletions in fetuses with cardiac anomalies detected on ultrasound examination. RESULTS: Of 64 cases, 18 had visible chromosome anomalies. FISH testing for 22q11.2 deletion was performed on the remaining 46 cases, and five exhibited a 22q11.2 deletion. Three of the five had de novo deletions, one was maternally inherited, and one family declined testing. CONCLUSION: FISH analysis for 22q11.2 deletion should be performed on all fetuses with cardiac defects (excluding hypoplastic left heart and echogenic focus) and a normal G-banded karyotype.

STIM1: a novel phosphoprotein located at the cell surface.

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.

Frequency and clinical significance of the S1235R mutation in the cystic fibrosis transmembrane conductance regulator gene: results from a collaborative study.

More than 900 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been reported to the cystic fibrosis (CF) consortium. A missense mutation, S1235R, was originally reported in a CF patient with a second mutation (G628R) on the same chromosome. The clinical significance of S1235R was not clear. S1235R is not among the commonly reported mutations, and it is not routinely screened for in most laboratories. However, we have detected the S1235R allele at a frequency that is significantly higher than that of many other CF mutations. Among more than 3,000 patients tested for either a possible diagnosis of CF or to determine CF carrier status, we identified 51 patients heterozygous for S1235R. No patients were homozygous for S1235R. Five patients were compound heterozygotes for a second CFTR mutation: two cases (one family) were N1303K/S1235R and three unrelated cases were deltaF508/S1235R. Our data suggest that S1235R, when combined with a second CF mutation, may be pathogenic, although phenotypic manifestations appear to be variable. The possibility that this represents a rare polymorphism cannot be discounted completely. Genetic counseling is difficult when S1235R is identified, even in the presence of a second known mutation, especially in prenatal cases.

Dual posttranscriptional targets of retinoic acid-induced gene expression.

Retinoic acid-induced differentiation of the pre-osteoblastic cell line, UMR 201, is associated with a marked increase in the proficiency of posttranscriptional nuclear processing of alkaline phosphatase mRNA. In this study we attempted to correlate the posttranscriptional actions of retinoic acid with changes in phosphorylation, or abundance of spliceosome components, or both. Treatment with retinoic acid for periods of < or = 4 h resulted in dephosphorylation of nuclear U1 70K protein without affecting its abundance. Peptide mapping showed that U1 70K dephosphorylation was related to the disappearance of one specific phosphopeptide out of four major U1 70K phosphopeptides. A twofold decrease in mRNA expression of an isoform of alternative splicing factor that inhibits splicing was also observed over the same period. Tumor necrosis factor-alpha, which enhances the posttranscriptional action of retinoic acid, reduced U1 70K mRNA expression, while an inhibition of retinoic acid action by transforming growth factor-beta was associated with a marked increase in U1 70K mRNA levels. Our results draw attention to the complex interactions between short- and long-term alterations in the abundance and functional status of U1 70K, as well as SR proteins by growth and/or differentiation factors in the regulation of spliceosome formation and function.

Cloning and molecular analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3.

Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer (poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]) consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), beta-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed by phbBPs-phbAPs-phbCPs. Upstream from phbBPs was found the phbRPs gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbRPs gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbRPs in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of beta-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBACPs in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 and phaDPs), and the gene order was ORF1, phaC1Ps, phaZPs, phaC2Ps, and phaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.

Transcriptional and posttranscriptional regulation of osteopontin gene expression in preosteoblasts by retinoic acid.

This study examines the relative importance of transcriptional and posttranscriptional actions of retinoic acid (RA) in the regulation of osteopontin gene expression in a rat clonal preosteoblastic cell line, UMR 201. Nuclear run-on analysis demonstrated constitutive expression of the osteopontin gene which was increased by threefold after 4 hr treatment with 1 microM RA, returning to a basal level by 24 hr. However, Northern blot analysis, performed concurrently, showed that RA progressively increased the steady-state osteopontin mRNA level beginning 2 hr before any increase in gene transcription and peaking at 24 hr. There was no difference in osteopontin mRNA stability between control and RA-treated cells after gene transcription was inhibited with 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB). Total RNA was obtained from cellular subfractions (nuclear matrix, nonmatrix chromatin, nuclear membrane, and cytoplasm) and reverse transcription-polymerase chain reaction (RT-PCR) performed with primers complementary to exons 3 and 4 of the mouse osteopontin gene. Unspliced PCR product, comprising the two exons and the intervening intron, was present in the nuclear matrix fractions of control and RA-treated cells. However, RA resulted in a time-dependent accumulation of mature osteopontin mRNA in all cellular subfractions, suggesting that the proficiency of nuclear processing of primary mRNA transcripts was greatly enhanced by RA. This action depended on de novo protein synthesis. These results demonstrate that the posttranscriptional action of RA is not unique to the regulation of alkaline phosphatase gene expression.

The impact of horizontal mergers and acquisitions on cost and quality in health care.

Mergers and acquisitions among HMOs, hospitals and other health care providers can be disconcerting to benefits staff and employees, but they can be successfully managed. They may offer an employer the opportunity to improve the quality of care provided and to do so at reduced costs.

Tumor necrosis factor alpha facilitates nuclear actions of retinoic acid to regulate expression of the alkaline phosphatase gene in preosteoblasts.

This study examines the molecular mechanisms of interaction between tumor necrosis factor alpha (TNF alpha) and retinoic acid on the expression of the alkaline phosphatase gene by rat clonal preosteoblastic cells. In this cell line, alkaline phosphatase mRNA was not constitutively expressed but was progressively induced by treatment with 1 microM retinoic acid, detectable by 6 h. Combining retinoic acid with 0.6 nM TNF alpha resulted in alkaline phosphatase mRNA appearing by 2 h, as well as enhanced expression above that observed with retinoic acid alone at 6, 12, and 24 h. Nuclear run-on analysis showed constitutive transcription of the alkaline phosphatase gene in control and TNF alpha-treated cells. At 4 h, retinoic acid, alone or combined with TNF alpha, increased alkaline phosphatase gene transcriptional rate by 2-fold. However, at 24 h, while no retinoic acid effect was retained, retinoic acid plus TNF alpha resulted in a 5-fold increase in alkaline phosphatase transcriptional rate. Examination of the distribution of nuclear alkaline phosphatase mRNA demonstrated that pre-spliced precursor mRNA was localized to the nuclear matrix in control and all treatment groups. Retinoic acid caused a time-dependent accumulation of mature, spliced alkaline phosphatase mRNA located in the non-matrix and cytoplasmic fractions, implying a post-transcriptional action of retinoic acid in nuclear processing and nucleocytoplasmic transport. Adding TNF alpha with retinoic acid greatly enhanced this effect, which was observed after 4 h, prior to any detectable interaction between TNF alpha and retinoic acid on gene transcription. In sharp contrast, only a negligible amount of nuclear processing occurred in control and TNF alpha-treated cells. This study reveals distinct interactions between TNF alpha and retinoic acid at post-transcriptional as well as transcriptional levels to regulate expression of the alkaline phosphatase gene in preosteoblasts.

Regulation and regulatory role of the retinoids.

Retinoids regulate differentiation and cellular growth, exerting their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs), and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Although RAR/RXR heterodimers form preferentially in vitro and in vivo, it does not exclude the possibility that RXR/RXR homodimers may regulate a distinct signaling pathway. Synthetic retinoids that selectively activate or antagonize retinoic acid receptor isoforms promises to be useful tools for the elucidation of specific retinoid response pathways. Considerable progress has been made in understanding the molecular basis underlying limb bud formation, particularly the manner in which retinoic acid interacts with other signaling molecules to determine pattern formation. The phenotypic abnormalities observed in compound null mutants of retinoid receptors, recapitulating those described in the vitamin A deficiency syndrome, confirm the crucial function of endogenous retinoids in fetal development. However, the absence of phenotypic abnormalities in null mutants of individual RAR isoforms raises the possibility of functional redundancy among RAR subtypes and at the same time challenging the concept that the diverse effects of retinoids are related to the multiplicity of fnctionally distinct receptors.

Novel action of retinoic acid. Stabilization of newly synthesized alkaline phosphatase transcripts.

Several observations led us to investigate the possibility that retinoic acid achieved its marked induction of alkaline phosphatase gene expression through a post-transcriptional effect in the nuclei of clonal rat pre-osteoblastic UMR 201 cells. The steady-state level of alkaline phosphatase mRNA was significantly stimulated by retinoic acid. Although nuclear run-on analysis showed that 10(-6) M retinoic acid caused an increase in alkaline phosphatase gene transcription, this was transient compared with the rise in alkaline phosphatase mRNA which continued to accumulate for many hours after retinoic acid stimulation of gene transcription had ceased. Moreover, the modest increase in transcriptional rate (approximately 2-fold) was not sufficient to account for the magnitude of the rise in mRNA level. In order, therefore, to examine the influence of retinoic acid on nuclear processing events, a cellular subfractionation method was applied. By nuclease protection analysis, and also by using reverse transcription-polymerase chain reaction, sequences corresponding to intron 2 and intron 4, respectively, were demonstrated specifically in the nuclear matrix fraction of both control and retinoic acid-treated cells. Mature (spliced) alkaline phosphatase mRNA accumulated in the non-matrix (DNase I/salt eluate, nuclear membrane) and cytoplasmic fractions of retinoic acid-treated cells at more than 100-fold greater levels than in control cells. This implies that nuclear processing of the primary RNA transcript occurred only in cells treated with retinoic acid. The post-transcriptional action of retinoic acid was inhibited by cotreatment with 10 micrograms/ml cycloheximide. Transforming growth factor beta (TGF beta) (1 ng/ml) did not influence whole cell alkaline phosphatase levels in UMR 201 cells. Nevertheless, TGF beta increased the transcriptional rate of the alkaline phosphatase gene. Although precursor mRNA was detected in the nuclear matrix fraction of TGF beta-treated cells, there was no evidence of further mRNA nuclear processing. The data are consistent with stabilization of nascent alkaline phosphatase mRNA chains by retinoic acid treatment and suggests that regulation of mRNA processing can be independent of gene transcription. This study demonstrates a novel post-transcriptional action of retinoic acid which plays an important, if not a dominant role, in determining the steady-state level of alkaline phosphatase mRNA.

Peritoneal dialysis for acute renal failure: overfeeding resulting from dextrose absorbed during dialysis.

Peritoneal dialysis is a relatively safe and effective form of therapy for acute renal failure (ARF). As dextrose in the dialysate provides the osmotic gradient to achieve fluid removal, frequent exchanges with dialysate containing high dextrose is occasionally used to achieve negative balance in fluid overloaded patients. It has previously been shown that dextrose absorption from the peritoneal cavity is significant. Using indirect calorimetry and analyzing the dialysate effluent for its dextrose concentration, we studied the effects of high dextrose-containing dialysate in five patients with ARF. Despite minimal intake of calories, all patients had an RQ greater than 1.0 consistent with net lipogenesis resulting from dextrose absorbed from the peritoneal cavity. Four of five patients absorbed greater than 500 g of dextrose over 24 h. As overfeeding could lead to hepatic steatosis, increased CO2 production with worsening of respiratory failure, and hyperglycemia, the risks of using high dextrose-containing dialysate fluids should be weighed carefully against potential benefits. When nutritional support is indicated in such patients, contribution of dextrose calories from dialysate fluid should be taken into account.

Opposing influences of glucocorticoid and retinoic acid on transcriptional control in preosteoblasts.

UMR 201 is a nontransformed rat clonal cell line derived from neonatal calvaria with phenotypic characteristics of preosteoblasts. Retinoic acid strongly induces expression of alkaline phosphatase and its mRNA in these cells. Dexamethasone substantially reduced the retinoic acid-induced expression of alkaline phosphatase. This apparent interaction between dexamethasone and retinoic acid effects raised the possibility that interactions may extend to other osteoblast-related phenotypic characteristics in UMR 201 cells. Treatment with dexamethasone resulted in a decrease in the expression of mRNA for pro-alpha 1(I) collagen, but upon coincubation with 1 microM retinoic acid for 24 h, the decrease in mRNA for pro-alpha 1(I) collagen was abrogated. Dexamethasone (Dex) treatment caused a dose-dependent increase in osteonectin mRNA, half maximally effective between 1 nM and 10 nM Dex. One micromolar of retinoic acid alone led to a small increase in expression of osteonectin mRNA but prevented any further increase when Dex was added to retinoic acid-treated cells. To study transcriptional control, osteonectin genomic fragments were linked to the bacterial reporter gene, chloramphenicol acetyltransferase, and introduced by transfection into UMR 201 cells. Dexamethasone increased the transcriptional activity of an osteonectin-chloramphenicol acetyltransferase construct; 100 nM Dex resulted in a 3-fold increase over control cells which was attenuated when 1 microM retinoic acid was added to the incubation, while retinoic acid alone resulted in a 2-fold increase in transcriptional activity. Finally, it was noted that coincubation with retinoic acid and Dex stimulated the proliferation of UMR 201 cells when compared with either treatment alone. This study shows the potential importance of hormonal interactions in the expression of osteoblast function.

Retinoic acid and tumour necrosis factor-alpha act in concert to control the level of alkaline phosphatase mRNA.

Retinoic acid has a specific role in cellular differentiation and is believed to act by regulating the transcription of specific genes. In the present work, evidence is provided to show that alkaline phosphatase (ALP) gene expression is mediated by retinoic acid in a model clonal cell line (UMR 201) derived from rat neonatal calvaria. These cells have the characteristics of relatively undifferentiated mesenchymal cells with a very low basal ALP activity which is dramatically increased by retinoic acid. Messenger RNA for ALP was clearly demonstrated when the cells were treated with 1 microM retinoic acid for 24 h. Recombinant human tumour necrosis factor-alpha (recombinant TNF-alpha) interacted with retinoic acid to potentiate the rise in ALP activity, although recombinant TNF-alpha alone had no effect. The potentiation of retinoic acid-induced ALP activity was correlated with an increased amount of mRNA for ALP with the combined treatment. By observing the rate of decay of mRNA for actin and ALP, we were able to demonstrate that the interaction between retinoic acid and recombinant TNF-alpha modulated the steady state of ALP mRNA. The mode of action of recombinant TNF-alpha may serve as a model for other paracrine regulators of cell function.