Phosphate effects in the fermentation of α-amylase by Bacillus amyloliquefaciens

Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding.     

Heat generation in laser irradiated tissue

Many medical applications involving lasers rely upon the generation of heat within the tissue for the desired therapeutic effect. Determination of the absorbed light energy in tissue is difficult in many cases. Although UV wavelengths of the excimer laser and 10.6 microns wavelength of the CO2 laser are absorbed within the first 20 microns of soft tissue, visible and near infrared wavelengths are scattered as well as absorbed. Typically, multiple scattering is a significant factor in the distribution of light in tissue and the resulting heat source term. An improved model is presented for estimating heat generation due to the absorption of a collimated (axisymmetric) laser beam and scattered light at each point r and z in tissue. Heat generated within tissue is a function of the laser power, the shape and size of the incident beam and the optical properties of the tissue at the irradiation wavelength. Key to the calculation of heat source strength is accurate estimation of the light distribution. Methods for experimentally determining the optical parameters of tissue are discussed in the context of the improved model.     

Identification of an unusual deletion within homologous repeats of human reticulocyte beta-spectrin and probable peptide polymorphism

We screened two different human reticulocyte cDNA libraries with beta-spectrin(beta Sp)-specific polyclonal antibodies and with our original radiolabeled human BSP cDNA probe (encoding beta Sp). Of the 20 independent clones, the largest had about a 2.5-kb insert corresponding to the deduced amino acid (aa) sequence of the beta-7 to beta-14 repetitive segments. Among these segments, segment 12 was 7 aa shorter than the other repetitive segments. We showed that this truncation was not a result of (i) cloning artifact, (ii) alternate splicing, or (iii) common genomic polymorphism by additional examination of 14 individual human chromosomes. Recently, another laboratory described the BSP nucleotide (nt) sequence overlapping partially with our sequence. These overlapping sequences were homologous with the exception of two nt differences at the positions 1342 and 1514. The discrepancy at nt 1342 changes the His to Arg. This newly derived probe has been used to find an additional example of BSP restriction fragment length polymorphism.     

Genotype of Yupik Eskimos with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Summary An A-to-G transition in the second intron was the sole mutation detected in four Yupik Eskimo patients with salt-wasting congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. Allele-specific hybridization should be an efficient means of performing prenatal diagnosis of the disease in this highly inbred population.     

Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines.

Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.     

Dideoxy fingerprinting (ddE): a rapid and efficient screen for the presence of mutations.

We describe dideoxy fingerprinting (ddF), a hybrid between dideoxy sequencing and SSCP that can detect the presence of single base and other sequence changes in PCR-amplified segments. As implemented herein, ddF involves a Sanger sequencing reaction with one dideoxynucleotide followed by nondenaturing gel electrophoresis. When ddF was used to examine segments of the human factor IX gene, 84 of 84 different mutations were detected with a very low rate of false positive signals. The approximate locations of the sequence changes could be determined from the ddF pattern and samples with different sequence changes had different fingerprints. In addition, large segments could be amplified and rapidly screened by ddF in multiple smaller subsegments. The patterns observed with ddF are instructive in that they suggest an inherent limitation in the detection of certain mutations by SSCP.     

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].