Assessing ureolytic bacteria with calcifying abilities isolated from limestone caves for biocalcification.

Biocalcification through the use of ureolytic bacteria and biochemical activities has evolved in recent decades into a fervent resourceful effective technology suitable for soil stabilization, crack repair and bioremediation. Extensive studies have been carried out on numerous ureolytic bacterial species isolated from soils and sewage samples. However, very limited attention has been given to limestone caves with natural calcite formations as a possible source for isolation of ureolytic bacteria. In this study, bacterial isolates were recovered from limestone cave samples to determine their suitability for biocalcification. Twenty-seven morphologically distinct bacterial isolates were identified by partial 16S rRNA gene sequencing and their various genetic diversity was characterized according to their phylogenetic affiliations. Based on the molecular identification, Sporosarcina was the most abundant genus among all the ureolytic isolates, while the rest belonged to Pseudogracilibacillus and Bacillus genera. Analytical analysis on urease measurement showed that urease activities for the isolates ranged from 1·130 to 21·513 mol urea hydrolysed per minute, with isolate NB33 achieving the highest value and TSB4 achieving the lowest value. The estimated CaCO3 precipitates for the isolates ranged from 4·04 to 17·26 mg ml-1 , with isolate NB30 achieving the highest value and TSB20 achieving the lowest value. The findings in this study demonstrated that the ureolytic bacteria from limestone caves are promising bio-calcifying agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Ureolytic bacteria continues to play an important role as microbial tools used in geotechnical engineering for soil biocalcification. Microbial strains with the ability to produce urease enzyme and induce calcium carbonate mineral are often isolated from soil, water and sludge samples. However, screening for these essential microbes from extreme regions such as caves are rarely investigated. In this study, native bacteria which were isolated from limestone cave samples are identified and characterized. The findings suggested that these ureolytic bacterial isolates have the potential to serve as suitable alternative microbial agents for soil strengthening and stabilization.

Inflammatory bowel disease cancer surveillance in a tertiary referral hospital: attitudes and practice.

BACKGROUND: Physician adherence to guidelines for colorectal cancer (CRC) surveillance in inflammatory bowel disease (IBD) is often poor. This may lead to adverse patient outcomes and excess endoscopic workload. AIMS: To assess the attitudes and practice of IBD specialists in a tertiary centre towards colonoscopic surveillance. METHODS: First, a questionnaire evaluating attitudes and approach to CRC surveillance was issued to 36 clinicians at one tertiary referral hospital. Second, a retrospective audit of IBD surveillance colonoscopy practice over a 2-year period was performed. RESULTS: Questionnaire response rate was 97%. Sixty-nine per cent of respondents were aware of, and used, Australian guidelines. Surveillance was undertaken by all clinicians in patients with extensive colitis, 83% in patients with left-sided colitis and 51% in patients with proctitis. Seventy-six per cent used chromoendoscopy, and 47% took 10 to 20 random biopsies. Colectomy was considered appropriate in 0% for unifocal low-grade dysplasia, 35% for multifocal low-grade dysplasia and 83% for high-grade dysplasia. Sixty-six per cent would remove elevated dysplastic lesions endoscopically. The audit identified 103 surveillance colonoscopies in 81 patients. Chromoendoscopy was used in 21% of cases, and the median number of random biopsies was 13. Sixty-two per cent of colonoscopies were performed outside the guidelines in relation to colonoscopic frequency. Following colonoscopy, an appropriate recommendation for subsequent surveillance was documented in 40% of cases. CONCLUSIONS: Knowledge and practice of CRC surveillance in IBD vary among specialist clinicians and often deviate from guidelines. Many clinicians perform surveillance earlier and more frequently than recommended. These findings have implications for patient outcomes and workload.

Retinoic acid biosynthesis by normal human breast epithelium is via aldehyde dehydrogenase 6, absent in MCF-7 cells.

Retinoic acid (RA) is the form of vitamin A that controls differentiation and proliferation of epithelia. Our previous work established that normal breast epithelia synthesize RA from retinol, an ability retained by three immortalized but nontumorigenic cell lines but lost in five of six breast cell lines. In this work, we characterize the cause of this defect in one of the lines, the MCF-7 line. We have determined that the immortalized but nontumorigenic cell line, MTSV1.7, capable of synthesizing RA from both retinol and retinal, contains a retinaldehyde dehydrogenase activity for the second step in RA biosynthesis. We have identified it, after isolation, as a previously described enzyme, aldehyde dehydrogenase 6 (ALDH6). Immunohistochemical analysis of normal human breast with antibodies to ALDH6 showed expression of this enzyme in the glandular epithelia colocalized with cellular RA-binding protein type II, a possible marker for certain cells able to synthesize RA. ALDH6 was not present in MCF-7 cells, and these cells were unable to oxidize retinal to RA in culture. When MCF-7 cells were then transfected with ALDH6, they (re)gained the ability to oxidize retinal to RA as well as some ability to synthesize RA when provided with retinol. This suggests that loss of ALDH6 expression is the defect in RA biosynthesis in these cells. Identification of ALDH6 as the retinaldehyde dehydrogenase present in normal human breast epithelia provides the first tool necessary for studying the loss of RA synthetic ability in cancer cells and the relationship of this process to malignant transformation.

Gene duplication gives rise to a new 17-kilodalton lipocalin that shows epididymal region-specific expression and testicular factor(s) regulation.

Using transgenic mice, we have recently shown that 5 kb of the 5'-flanking region of the mouse epididymal retinoic acid-binding protein (mE-RABP) gene contains all of the information required for spatial and temporal gene expression in the epididymis. To identify the important cis-DNA regulatory element(s) involved in the tissue-, region-, and cell-specific expression of the mE-RABP gene, the 5-kb DNA fragment was sequenced. A computer analysis of the nucleotide sequence showed the presence of a new gene located 1.7 kb upstream from the mE-RABP gene transcription initiation site. The analysis of the open reading frame showed that the new gene encoded a putative 17-kDa lipocalin (named mEP17) related to mE-RABP. A 600-bp complementary DNA encoding mEP17 was cloned by rapid amplification of 3'-cDNA ends from epididymal total RNA. Two mEP17 RNA species (1 and 3.1 kb in size) were detected by Northern blot in the epididymis, but not in other tissues tested. In situ hybridization analyses showed that, unlike mE-RABP messenger RNA (mRNA), which is expressed in the distal caput epididymidis, mEP17 mRNA was detected only in the principal cells of the initial segment. The spatial expression and homology with mE-RABP suggest that mEP17 may act as a retinoid carrier protein within the epididymis. mEP17 mRNA expression disappeared 5 days postcastration. Four days after unilateral castration, mEP17 mRNA had nearly disappeared in the epididymis from the castrated side, but not from the intact side. In addition, testosterone replacement to bilaterally castrated mice failed to restore gene expression. We conclude that mEP17 gene expression is dependent on testicular factors circulating in the luminal fluid. Together our results suggest that mE-RABP and mEP17 genes were generated by duplication and that evolution led to a different region-specific gene expression and regulation in the epididymis.

Plasma retinol binding protein: structure and function of the prototypic lipocalin.

In terms of both structure and biological function, retinol binding protein (RBP) is one of the best characterized members of the lipocalin superfamily. The molecular interactions in which RBP participates are described herein.

Retinol conversion to retinoic acid is impaired in breast cancer cell lines relative to normal cells.

The bioactivity of retinol (vitamin A) is in part dependent on its metabolism to retinoic acid (RA). We investigated the ability of breast epithelial cells to synthesize RA when challenged with a physiological retinol dose (2 microM). Normal human mammary epithelial cells (HMEC) cultured from reduction mammoplasties were competent in RA synthesis and the ability to synthesize RA was retained by immortal, nontumorigenic breast epithelial cell lines (MTSV1.7, MCF-10F, and 184B5). In contrast, most (five of six) breast cancer cell lines could not synthesize RA or did so at low rates relative to normal cells. A notable exception was the MDA-MB-468 cell line, which was fully competent in RA synthesis. Most (>/=68%) of the RA synthesized by breast cells was recovered from the culture medium. Cellular retinol binding protein and cellular RA binding protein II, both expressed in HMEC, had various expression patterns in the cell lines that did not correlate with the observed differences in RA synthesizing ability. Strong RA induction of the RA hydroxylase P450RAI (CYP26) was confined to ERalpha-positive T47D and MCF-7 breast cancer cells and did not appear to explain the lack of detectable RA levels in these cells since RA remained undetectable when the cells were treated with 5-10 microM liarozole, a P450RAI inhibitor. We hypothesize that retinol bioactivity is impaired in breast cancer cells that cannot synthesize RA. In preliminary support of this hypothesis, we found that retinol (0.5-2 microM) inhibited MCF-10F but not T47D or MCF-7 cell growth.

Retinoic acid synthesis and expression of cellular retinol-binding protein and cellular retinoic acid-binding protein type II are concurrent with decidualization of rat uterine stromal cells.

Decidualization of stromal cells at the site of embryo implantation in the rat uterus is accompanied by expression of cellular retinol-binding protein and cellular retinoic acid-binding protein [CRABP(II)], whose presence has been shown to correlate with gain of ability to synthesize retinoic acid in other cells. Here we examined whether decidual cells also acquired the ability to synthesize retinoic acid, which would have important implications for understanding the implantation process. Decidual cells were isolated from the uterus on day 8 of pregnancy and cultured. When provided with retinol, they indeed synthesized and released retinoic acid to the medium. To follow acquisition of this ability more closely, artificial induction of decidualization was exploited. Ovariectomized rats were placed on a hormonal regimen that allows decidualization to occur in vivo, with oil stimulation, or in vitro, if cells are isolated on day 5 of the regimen and then cultured. Decidualization in vivo reproduced the expression of cellular retinol-binding protein and CRABP(II) seen during pregnancy. Stromal cells isolated on regimen day 2 synthesized little retinoic acid and expressed little alkaline phosphatase, a marker of decidualization. Stromal cells isolated on regimen day 5 had elevated levels of alkaline phosphatase, increasing during the 3 days of culture examined. The ability of the stromal cells to synthesize retinoic acid showed the same pattern: a substantially elevated production from that previously observed, on day 2, with production increasing significantly over the next 2 culture days. Thus, expression of CRABP(II) was correlated with gain of ability to synthesize retinoic acid. Retinoid signaling may be an important part of the process of embryo implantation.

Cellular retinoic acid binding protein is associated with mitochondria.

We report that immunohistochemical staining for cellular retinoic acid-binding protein (CRABP) was restricted to the cytoplasm of cortical cells in bovine adrenal. In contrast, staining for the similar protein, cellular retinol-binding protein (CRBP), was found throughout these cells. After transfections of CRABP and CRBP into cultured cells, immunofluorescence analyses again revealed cytoplasmic restriction only for CRABP, with a pronounced punctate appearance. Use of organelle-specific fluorochromes indicated that CRABP immunofluorescence overlaid exactly with the pattern of the mitochondrial-specific fluorochrome. Confirmation of this association came with subcellular fractionation of the adrenal cortex. CRABP, but not CRBP, co-sedimented with the mitochondria, a novel finding for a member of this superfamily of cellular lipid-binding proteins.

Vitamin A-sensitive tissues in transgenic mice expressing high levels of human cellular retinol-binding protein type I are not altered phenotypically.

The suggested function of cellular retinol-binding protein type I [CRBP(I)] is to carry retinol to esterifying or oxidizing enzymes. The retinyl esters are used in storage or transport, whereas oxidized forms such as all-trans or 9-cis retinoic acid are metabolites used in the mechanism of action of vitamin A. Thus, high expression of human CRBP(I) [hCRBP(I)] in transgenic mice might be expected to increase the production of retinoic acid in tissues, thereby inducing a phenotype resembling vitamin A toxicity. Alternatively, a vitamin A-deficient phenotype could also be envisioned as a result of an increased accumulation of vitamin A in storage cells induced by a high hCRBP(I) level. Signs of vitamin A toxicity or deficiency were therefore examined in tissues from transgenic mice with ectopic expression of hCRBP(I). Testis and intestine, the tissues with the highest expression of the transgene, showed normal gross morphology. Similarly, no abnormalities were observed in other tissues known to be sensitive to vitamin A status such as cornea and retina, and the epithelia in the cervix, trachea and skin. Furthermore, hematologic variables known to be influenced by vitamin A status such as the hemoglobin concentration, hematocrits and the number of red blood cells were within normal ranges in the transgenic mice. In conclusion, these transgenic mice have normal function of vitamin A despite high expression of hCRBP(I) in several tissues.

A 5-kilobase pair promoter fragment of the murine epididymal retinoic acid-binding protein gene drives the tissue-specific, cell-specific, and androgen-regulated expression of a foreign gene in the epididymis of transgenic mice.

The murine epididymis synthesizes and secretes a retinoic acid-binding protein (mE-RABP) that belongs to the lipocalin superfamily. The gene encoding mE-RABP is specifically expressed in the mouse mid/distal caput epididymidis under androgen control. In transgenic mice, a 5-kilobase pair (kb) promoter fragment, but not a 0.6-kb fragment, of the mE-RABP gene driving the chloramphenicol acetyltransferase (CAT) reporter gene restricted high level of transgene expression to the caput epididymidis. No transgene expression was detected in any other male or female tissues. Immunolocalization of the CAT protein and in situ hybridization of the corresponding CAT mRNA indicated that transgene expression occurred in the principal cells of the mid/distal caput epididymidis, thereby mimicking the spatial endogenous mE-RABP gene expression. Transgene and mE-RABP gene expression was detected from 30 days and progressively increased until 60 days of age. Castration, efferent duct ligation, and hormone replacement studies demonstrated that transgene expression was specifically regulated by androgen but not by any other testicular factors. Altogether, our results demonstrate that the 5-kb promoter fragment of the mE-RABP gene contains all of the information required for the hormonal regulation and the spatial and temporal expression of the mE-RABP gene in the epididymis.

Synthesis of retinoic acid by rat ovarian cells that express cellular retinoic acid-binding protein-II.

The induction of pseudopregnancy by the injection of eCG in rats results in the appearance of cellular retinoic acid-binding protein type II (CRABP[II]) in the granulosa cells of the ovary and the lining epithelium of the uterus within 48 h. This expression pattern is also seen in the normal mature female rat, in which CRABP(II) is expressed in the uterine epithelium during estrus (but not diestrus) and in the granulosa and luteal cells of the ovary. We have previously demonstrated that the uterine epithelial cells from the pseudopregnant rat have gained the ability to synthesize retinoic acid from retinol, in correlation with the induced expression of CRABP(II). If this is true for other sites of CRABP(II) expression, then local production of retinoic acid is intimately connected with various stages of reproduction in the female. Here we report that granulosa cells from the ovary of the eCG-treated immature rat and luteal cells from the ovary of the eCG/hCG-treated immature rat (both of which express CRABP[II]) synthesized markedly higher amounts of retinoic acid when cultured, compared to granulosa cells cultured from the ovary of the prepubertal rat treated with control vehicle. Culturing the granulosa cells from either control or eCG-treated animals had no effect on the expression of CRABP(II) cells. These data are consistent with our hypothesis that CRABP(II) expression is associated with retinoic acid synthesis and strengthen the case that local generation of retinoic acid plays an important role in reproduction.

Retinoid-binding proteins in the cerebellum and choroid plexus and their relationship to regionalized retinoic acid synthesis and degradation.

The expression of cellular retinoic-acid-binding protein (CRABP) and cellular retinol-binding protein (CRBP), as well as their relationship to retinoic acid (RA) synthesis and degradation were examined in the developing mouse cerebellum and choroid plexus of the fourth ventricle. The choroid plexus, which expresses the RA-synthesizing retinaldehyde dehydrogenase RALDH-2, is likely to represent a diffusion source of RA for the closely apposed cerebellum, regulating its development. We found CRBP to be expressed in the choroid plexus and, in an in-vitro assay, addition of recombinant CRBP to RALDH-2 increased RA synthesis from retinaldehyde, with the amount of increase depending on the CRBP/retinaldehyde ratio. A technique that characterizes RA-binding proteins according to their isoelectric point showed both CRABP I and CRABP II to be present in the cerebellum and P19 cells, and only CRABP II to be present in the choroid plexus. With this technique, CRABP I could also be detected in the HL60 cell line. In addition to the two known acidic RA-binding proteins CRABP I and II, the cerebellum expressed a third RA-binding protein distinguishable by its neutral isoelectric point; the same binding protein was also detected in the olfactory bulb, kidney and testes. We used the RA-binding technique to follow the rate of elimination of bound RA from the cerebellum. A systemic injection of 0.3 micromols RA into postnatal day-1 mice was almost completely removed after 8 hours. These results suggest mechanisms by which the retinoid-binding protein may regulate the equilibrium of RA synthesis and catabolism in the cerebellum and choroid plexus.

Genomic organization and chromosomal localization of the murine epididymal retinoic acid-binding protein (mE-RABP) gene.

The murine epididymal retinoic acid-binding protein (mE-RABP) is specifically synthesized in the mouse mid/distal caput epididymidis and secreted in the lumen. In this report, we have demonstrated by Southern blot analysis of genomic DNA that mE-RABP is encoded by a single-copy gene. A mouse 129/SvJ genomic bacterial artificial chromosome (BAC) library was screened using a cDNA encoding the minor form of mE-RABP. One positive BAC clone was characterized and sequenced to determine the nucleotide sequence of the entire mE-RABP gene. The molecular cloning of the mE-RABP gene completes the characterization of the 20.5-kDa-predicted preprotein leading to the minor and major forms of mE-RABP. Comparison of the DNA sequence of the promoter and coding regions with that of the rat epididymal secretory protein I (ESP I) gene showed that the mE-RABP gene is the orthologue of the ESP I gene that encodes a rat epididymal retinoic acid-binding protein. Several regulatory elements, including a putative androgen receptor binding site, "CACCC-boxes," NF-1, Oct-1, and SP-1 recognition sites, are conserved in the proximal promoter. Analysis of the nucleotide sequence of the mE-RABP gene revealed the presence of seven exons and showed that the genomic organization is highly related to other genes encoding lipocalins. The mE-RABP gene was mapped by fluorescent in situ hybridization to the [A3-B] region of the murine chromosome 2. Our data, combined with that of others, suggest that the proximal segment of the mouse chromosome 2 may be a rich region for genes encoding lipocalins with a genomic organization highly related to the mE-RABP gene.

Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid.

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.

Spatial and temporal patterns of expression of cellular retinol-binding protein and cellular retinoic acid-binding proteins in rat uterus during early pregnancy.

Retinoic acid, perhaps the most potent hormonal form of the naturally occurring retinoids (retinol and derivatives), is required in vivo for the maintenance of normal pregnancy and embryo development. However, little is known about the specific sites of action and metabolism in the uterus during pregnancy. In this study we describe the pattern of temporal and cell-specific expression of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding proteins type I and type II (CRABP and CRABP[II], respectively) in the rat uterus during the periimplantation period (Day 1 to Day 7 of pregnancy; Day 1 = presence of vaginal plug). Immunohistochemical studies showed that there were dramatic and rapid changes in expression pattern of the retinoid-binding proteins after mating as early as Day 1, as well as a differential expression of these proteins when the mesometrial side and antimesometrial side of the uterus were examined during the periimplantation period. CRABP(II), whose presence has been previously shown to correlate with retinoic acid synthesis in the uterine epithelium, was specifically localized to the luminal epithelium at Day 1, being stronger on the mesometrial side, and then fell to lower levels. CRBP was also expressed in the luminal epithelium on the mesometrial side at Day 1 as well as in some stromal cells, declining at these sites over the next several days. CRABP was localized to some of the stromal cells at Day 1, overlapping CRBP expression. Embryonic implantation was accompanied by the appearance of CRBP and CRABP(II) in the decidual cells. CRBP and CRABP were also present in both smooth muscle layers of the uterus. The changes in the temporal and cell-specific distribution of retinoid-binding proteins imply a multifunctional role of vitamin A in uterine cell proliferation, differentiation, and embryonic implantation. The presence of CRABP(II) suggests that local generation of retinoic acid is important in these processes.

Bovine epidermal fatty acid-binding protein: determination of ligand specificity and cellular localization in retina and testis.

The fatty acid-binding protein (FABP) family consists of small, cytosolic proteins believed to be involved in the uptake, transport, and solubilization of their hydrophobic ligands. Members of this family have highly conserved sequences and tertiary structures. Using an antibody against testis lipid-binding protein, a member of the FABP family, a protein was identified from bovine retina and testis that coeluted with exogenously added docosahexaenoic acid during purification. Amino acid sequencing and subsequent isolation of its cDNA revealed it to be nearly identical to a bovine protein expressed in the differentiating lens and to be the likely bovine homologue of the human epidermal fatty acid-binding protein (E-FABP). From quantitative Western blot analysis, it was estimated that bovine E-FABP comprised 0.9%, 0.1%, and 2.4% of retina, testis, and lens cytosolic proteins, respectively. Binding studies using the fluorescent probe ADIFAB indicated that this protein bound fatty acids of differing levels of saturation with relatively high affinities. Kd values ranged from 27 to 97 nM. In addition, the protein was immunolocalized to the Müller cells in the retina as well as to Sertoli cells in the testis. The location of bovine E-FABP in cells known to be supportive to other cell types in their tissues and the ability of E-FABP to bind a variety of fatty acids with similar affinities indicate that it may be involved in the uptake and transport of fatty acids essential for the nourishment of the surrounding cell types.

Structure and putative function of a murine epididymal retinoic acid-binding protein (mE-RABP).

Vitamin A is required to maintain the epididymal epithelium. In this report, the characterization and putative functions of a murine epididymal retinoic acid-binding protein (mE-RABP) that is secreted into the lumen from the mid-/distal caput epididymidis are discussed. The amino acid sequence analysis of the mE-RABP preprotein shows that mE-RABP is the mouse orthologue of the rat epididymal secretory protein I (ESPI). These proteins belong to the lipocalin superfamily and bind to active retinoids but not to retinol. Therefore, we propose that mE-RABP may function as an extracellular retinoid carrier-protein involved in the paracrine regulation of epididymal function by retinoids.