Production rates of Eurytemora affinis in the Elbe estuary,comparison of field and enclosure production estimates

Production rates of the calanoid copepod Eurytemora affinis were estimated from field studies in the Elbe estuary and from an enclosure experiment. As one basic parameter of production rates, the body length, was compared between both investigations. Most of the copepodid stages in the enclosure experiment reached a significant greater length than the copepodids in the estuary. The differences in length between copepods from the field and the experiment could mainly be explained by a four times higher chlorophyll-a level in the enclosure experiment. The better food supply also results in a higher individual growth rate for all instars in the enclosure experiment. Therefore the population of Eurytemora affinis in the Elbe estuary was regarded as food limited during certain times of the year, especially in late spring and summer.Maximum daily production rate in the enclosure experiment (40 µg dw l^–1 d^–1) was four times higher than in the estuary (12 µg dw l^–1 d^–1). The mean daily P:B ratio in the enclosure was 0.301 d^–1 compared to 0.11 d^–1 in the estuary.     

Phenotypic features of breast cancer cells overexpressing ornithine-decarboxylase

Polyamines (PA) have been shown to be critical mediators of estradiol-induced breast cancer cell proliferation. This finding suggests that constitutive activation of the PA pathway may promote tumor progression, possibly leading to hormone independence. To test this hypothesis, we transfected hormone-responsive MCF-7 breast cancer cells with a complementary DNA coding for ornithine-decarboxylase (ODC), the first rate-limiting enzyme in PA biosynthesis. Marked ODC over-expression observed in stably transfected clones was associated with a selective increase in cellular putrescine content, while spermidine and spermine levels were not altered. ODC-overexpressing MCF-7 cells were resistant to the antiproliferative effects of low but not high concentrations of the enzyme inhibitor, α-difluoromethylornithine. In agreement with our hypothesis, sensitivity to the growth-promoting action of estradiol was reduced by approximately one third (P < 0.001) in ODC-overexpressing MCF-7 cells compared with vector-only transfected clones. Basal growth under anchorage-dependent conditions was only marginally increased by ODC overexpression (P = 0.048), while clonogenicity in soft agar was actually reduced. These data suggest that activation of PA biosynthesis may contribute in part to the acquisition of estrogen independence by breast cancer cells. Since only putrescine content was increased as a result of ODC overexpression, these data may underestimate the overall influence of the PA pathway on breast cancer phenotype. © 1995 Wiley-Liss, Inc.     

Processing of secretogranin II by prohormone convertases: Importance ofPC1 in generation of secretoneurin

Secretoneurin is a recently characterized neuropeptidepresent in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.     

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture

The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA- containing SV40 ori and immunoglobulin gene for transiently expressing protein. The bcl–2 expressing COS–1 cells produced more protein than the mock transfected COS–1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.     

Metal-modified nucleobase pairs involving 7,9-dimethylguanine: trans-a2PtII analogues (a = NH3 or CH3NH2) of Watson-Crick GC and homo GG pairs

 The analogy between H-bonded nucleobase pairs and their metalated analogues is extended to the hemiprotonated pair of 7,9-dimethylguanine (7,9-DimeG) and the Watson-Crick and reversed Watson-Crick pair between 7,9-dimethylguaninium (7,9-DimeGH⁺) and 1-methylcytosine (1-MeC). The crystal structure analyses of two model compounds, trans–[Pt(CH₃NH₂)₂(7,9-DimeG-N1)₂](NO₃)₂ (1) and trans–[Pt(NH₃)₂(1-MeC-N3)(7, 9-DimeG-N1)](PF₆)₂· 2.5 H₂O (3a) are reported. Pt binding is through N1 of 7,9-DimeG and N3 of 1-MeC. In solution, 3a exists in a mixture with Watson-Crick and reversed Watson-Crick arrangements of the two bases, depending on solvent, concentration and anions. Received: 16 October 1996 / Accepted: 27 January 1997     

Microquantification of proteins with low chromophore content

The advantages of the intrinsic fluorescence of the tryptophan and the absorbance of the methionine residues of the 18 kDa-hsp - a recombinant protein from Mycobacterium leprae - was exploited here to develop a sensitive and low costs method for protein assaying. They presented linearity between 3 and 1000 g of protein. The correlations between intrinsic fluorescence or absorbance at 230 nm and protein contents were both superiors to 0.99. These methods can be extended to others proteins with low aromatic residue contents.     

Expression of {beta}-galactoside {alpha}2,6-sialyltransferase does not alter the susceptibility of human colon cancer cells to NK-mediated cell lysis

The extent of processing of N-linked oligosaccharides and thesialylation of the target cell membranes has been positivelycorrelated with resistance to lysis mediated by NK cells, buta conclusive evidence has never been reached. Colon cancer tissuesexpress an increased activity of ß-ga-lactoside     

Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.     

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non- transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8- month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF- II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I