Propagation of Geotrichum candidum in Acid Brine

Geotrichum candidum completely neutralized the acid brine and reduced its biochemical oxygen demand (BOD) by 88%. Yield of dry mycelium was 62 g per 100 g of BOD utilized.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Pyrroline-5-carboxylate reductase from Cucurbita cotyledons

Pyrroline-5-carboxylate reductase, which required reduced pyridine nucleotide and Δ′-pyrroline-5-carboxylate for proline synthesis, was isolated from pumpkin cotyledons. The enzyme was found in the soluble fraction and had a 4.5-fold greater activity with NADH than NADPH. The enzyme was inhibited by NH₂OH, NADP, ATP and slightly by proline. Glutathione or pyridoxal-5-phosphate had little effect on enzyme activity. The enzyme had a pH optimum between 7·0 and 7·6 and was not inhibited by high concentrations of NADH or Δ′-pyrroline-5-carboxylate.     

Ornithine transaminase from Cucurbita maxima cotyledons

During germination a marked increase in both soluble and particulate ornthine transaminase occurs in pumpkin cotyledons. Both enzymes had a pH optimum of 8.3 and a requirement for ornthine and α-ketoglutarate. Other keto acids or amino donors showed little activity. The enzymes required an active sulphydryl group for maximum activity. Exogenous pyridoxal phosphate was not required, but hydroxylamine inhibited the reaction and added pyridoxal phosphate overcame this inhibition. Proline inhibited the reaction and may play a role in the fate of ornithine in pumpkin cotyledons.     

Studies on Media for Enumerating Enterococci in Frozen Vegetables

A study was made of the relative sensitivity and specificity of presumptive and confirmatory media for the most probable number enumeration of enterococci in frozen vegetables. Azide dextrose broth yielded the highest numbers of confirmable enterococci and its sensitivity was shown to be comparable to nonselective media. The use of ethyl violet azide broth as a confirmatory medium resulted in a significant number of false positive tests. Growth in broth containing 6.5% sodium chloride incubated at 45 C for 2 days was found to be a more specific confirmatory test for enterococci.     

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.      

The metabolism of proline in cotyledons of pumpkin (Cucurbita moschata)

Exogenous proline-U-₁₄C is readily metabolized to glutamate,ornithine, sugars, CO₂, and organic acids, and is incorporatedinto protein by etiolated and green pumpkin cotyledons. As littletranslocation of proline from the cotyledons occur, it was proposedthat in young tissue proline is converted to glutamate, ornithineor sugar which are then readily translocated from the cotyledons.In older tissue some glutamate carbon derived from proline isalso used as an energy source and metabolized to CO₂. As proteinsynthesis is occurring rapidly in these cotyledons, considerableproline is incorporated into new protein. After 10-hr, 15% ofthe absorbed radioactivity still remained as free proline. ¹Present address: Instituto de Ciencias Biologicas, UniversidadeFederal de Vicosa, Vicosa, Minas Gerais, Brasil. (Received February 1, 1974; )     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.