Transport of valine across the small intestinal epithelium in pigs fed different valine levels and Bacillus subtilis.

Mutants of Bacillus subtilis overproducing valine (B. subtilis VAL) could be an approach to supply pigs dietary valine (Val). In the study, 18 gilts were fed: (i) negative diet with a standardized ileal digestible (SID) Val:Lys of 0.63:1 (Neg); (ii) Neg added B. subtilis VAL (1.28 × 1011  cfu/kg as-fed) or; (iii) Neg added L-Val to a Val:Lys of 0.69:1. Using the Ussing chamber method, the study aimed to investigate whether (i) the diets affect intestinal transport of additions of 0, 5, 10 or 20 mmol Val/L from the mucosal to the serosal side and (ii) the B. subtilis VAL contributes to a net transport of Val produced in situ. The results showed that the Isc (ΔIscVal ) and release of Val to the serosal side solution (Srel ; µmol cm-2  min-1 ) increased with Val addition (linear and quadratic, p < .0001) but was similar for 5, 10 or 20 mmol Val/L and not affected by diet. No net transport of in situ produced Val by B. subtilis VAL was detected. In conclusion, feeding a Val-deficient diet with or without B. subtilis VAL or a Val sufficient diet did not affect the Val transport across intestinal epithelia. No in situ Val production by B. subtilis VAL was observed in the Ussing chambers.

Evaluation of in situ valine production by Bacillus subtilis in young pigs.

Mutants of Bacillus subtilis can be developed to overproduce Val in vitro. It was hypothesized that addition of Bacillus subtilis mutants to pig diets can be a strategy to supply the animal with Val. The objective was to investigate the effect of Bacillus subtilis mutants on growth performance and blood amino acid (AA) concentrations when fed to piglets. Experiment 1 included 18 pigs (15.0±1.1 kg) fed one of three diets containing either 0.63 or 0.69 standardized ileal digestible (SID) Val : Lys, or 0.63 SID Val : Lys supplemented with a Bacillus subtilis mutant (mutant 1). Blood samples were obtained 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding and analyzed for AAs. In Experiment 2, 80 piglets (9.1±1.1 kg) were fed one of four diets containing 0.63 or 0.67 SID Val : Lys, or 0.63 SID Val : Lys supplemented with another Bacillus subtilis mutant (mutant 2) or its parent wild type. Average daily feed intake, daily weight gain and feed conversion ratio were measured on days 7, 14 and 21. On day 17, blood samples were taken and analyzed for AAs. On days 24 to 26, six pigs from each dietary treatment were fitted with a permanent jugular vein catheter, and blood samples were taken for AA analysis 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding. In experiment 1, Bacillus subtilis mutant 1 tended (P<0.10) to increase the plasma levels of Val at 2 and 3 h post-feeding, but this was not confirmed in Experiment 2. In Experiment 2, Bacillus subtilis mutant 2 and the wild type did not result in a growth performance different from the negative and positive controls. In conclusion, results obtained with the mutant strains of Bacillus subtilis were not better than results obtained with the wild-type strain, and for both strains, the results were not different than the negative control.

Production of tissue plasminogen activator (t-PA) in Aspergillus niger.

A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.

The foldase CYPB is a component of the secretory pathway of Aspergillus niger and contains the endoplasmic reticulum retention signal HEEL.

Here we report the isolation and characterization of the cypB gene from Aspergillus niger. The cypB gene encodes a protein with a predicted molecular weight of 20.7 kDa, which shows a high degree of identity to the cyclophilin family of peptidyl prolyl cis-trans isomerases (PPIases) from other eukaryotes. The 5' untranslated region of cypB includes three sequences resembling UPREs (unfolded protein response elements). The expression of cypB is upregulated by tunicamycin and DTT, suggesting that at least one UPRE is functional. The CYPB protein also has a 23-amino acid sequence which serves to target the protein to the endoplasmic reticulum (ER), and the ER retention sequence HEEL. CYPB-(His)(6) was expressed in Escherichia coli; the purified protein is capable of isomerizing a substrate peptide in vitro. This is also the first report to show that C-terminal addition of the sequence HEEL is sufficient to ensure retention of the green fluorescent protein (GFP) within the ER.

The Aspergillus niger cypA gene encodes a cyclophilin that mediates sensitivity to the immunosuppressant cyclosporin A.

Here we report the cloning and characterization of a gene, cypA, from Aspergillus niger that encodes a peptidyl prolyl cis-trans isomerase (PPIase) belonging to the cyclophilin family. Sequencing of both genomic and cDNA clones revealed two ORFs in cypA, one encoding a 19-kDa protein of 174 amino acid residues and the other a 24-kDa protein of 219 amino acid residues, with overall identities of 27-77% to the homologous cyclophilins from prokaryotic and eukaryotic organisms. Expression of the 19-kDa CYPA-(His)(6) in E. coli shows that the purified protein has PPIase activity which is inhibited by cyclosporin A. Northern analysis shows two specific cypA transcripts, the smaller transcript encodes the cytosolic 19-kDa CYPA protein, the larger transcript encodes the putative mitochondrial 24 kDa CYPA protein. The transcript for the cytosolic CYPA is expressed at a higher basal level than that for the mitochondrial protein. The presence of tunicamycin, DTT or cyclosporin A in the medium does not affect the expression level of cypA. Its expression is however slightly induced by heat shock. Growing A. niger mycelium in the presence of cyclosporin A leads to an increase in hyphal branching prior to growth arrest. Overexpression of cypA under the control of its own promoter in A. niger results in increased sensitivity to cyclosporin A, suggesting that cypA encodes the cellular target for cyclosporin A in A. niger.

Immunolocalization and quantification of noncollagenous bone matrix proteins in methylmethacrylate-embedded adult human bone in combination with histomorphometry.

The noncollagenous proteins (NCPs) in the bone matrix comprise growth factors with distinct cellular effects and a series of proteins with less clear biological actions. In order to understand the role of these proteins in bone metabolism and in bone diseases, it is crucial to determine their localization and quantity in normal and pathological bone. We have developed an immunohistochemical method to detect osteopontin, osteocalcin, bone sialoprotein, osteonectin, decorin, biglycan, and the growth factors transforming growth factor-beta, insulin-like growth factor-I, and bone morphogenetic protein-2 both in bone matrix and in bone cells of adult human bone embedded in methylmethacrylate. Immunohistochemistry and standard bone histomorphometry in adjacent sections allows the localization of the proteins to metabolically active sites in bone. The protocol works with several fixatives and with bone specimens obtained and embedded to over 20 years ago. Most importantly, we developed a procedure to specifically stain the mineralized matrix green in combination with a red staining of the NCPs. Using digital image analysis it is possible to quantify the relative amounts of NCPs (microm2 NCP area/microm2 mineralized matrix area). Within one biopsy of normal bone cut at four different heights (at a distance of 100 microm), two adjacent sections were stained either for osteopontin or osteonectin. Thirty trabecular and 20 cortical microscopic fields were measured, and the NCP:mineralized matrix ratio was calculated. Stepwise analysis of the standard error of the mean of the NCP:mineralized matrix ratios showed that measuring about 50 microscopic fields is sufficient to obtain representative data with a small confidence interval. In conclusion, the present procedure enables to quantify NCPs and to relate their presence to metabolically active sites in bone. The quantification provides the opportunity to monitor differences in distribution (e.g., cortical vs. trabecular) and differences between normal and pathological conditions and to assess changes in matrix composition during treatment. This can be done by reanalyzing bone biopsies obtained in the past, e.g., during clinical trials. Therefore, the present technique will be a valuable tool for the study of noncollagenous bone matrix proteins in human bone.

A thionin stain for visualizing bone cells, mineralizing fronts and cement lines in undecalcified bone sections.

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored in the dark at 7 C.