Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiotrichum curvatum

Summary From six unsaturated fatty acid auxotrophs (Ufa mutants) of the oleaginous yeast Apiotrichum curvatum blocked in the conversion of stearic to oleic acid, were isolated revertants able to grow in the absence of unsaturated fatty acids, in a search for strains that can produce cocoa butter equivalents. A broad range in the percentage of saturated fatty acids (%SFA) was observed in the lipids of individual revertants (varying from 27%–86% SFA), compared with the wild-type (44% SFA). Further analysis of fatty acid composition indicated that: (i) not all six Ufa mutants had the same genetic background and (ii) one specific Ufa mutation could be reverted in more than one way. Revertants that produced lipids with a %SFA>56%, were examined further. These strains were cultivated for 50 generations and half of them produced lipids with high %SFA after that time and were defined as stable. The viability of revertant strains with extremely high %SFA (>80%) may be explained by our finding that polar lipids, which are part of yeast membranes, contained much more polyunsaturated fatty acids and a significantly lower %SFA than neutral (storage) lipids. One revertant (R25.75) was selected that was able to produce lipids in whey permeate at a rate comparable with wild-type A. curvatum and with a fatty acid composition and congelation curve comparable with cocoa butter. Offprint requests to: A. Ykema     

DEVELOPMENTAL STABILITY IN LEAVES OF CLARKIA TEMBLORIENSIS (ONAGRACEAE) AS RELATED TO POPULATION OUTCROSSING RATES AND HETEROZYGOSITY

Four natural populations of Clarkia tembloriensis, whose levels of heterozygosity and rates of outcrossing were previously found to be correlated, are examined for developmental instability in their leaves. From the northern end of the species range, we compare a predominantly selfing population (t? = 0.26) with a more outcrossed population (t? = 0.84), which is genetically similar. From the southern end of the range, we compare a highly selfing population (t? = 0.03) with a more outcrossed population (t? = 0.58). We measured developmental stability in the populations using two measures of within-plant variation in leaf length as well as calculations of fluctuating asymmetry (FA) for several leaf traits. Growth-chamber experiments show that selfing populations are significantly more variable in leaf length than more outcrossed populations. Developmental instability can contribute to this difference in population-level variance. Plants from more homozygous populations tend to have greater within-plant variance over developmentally comparable nodes than plants from more heterozygous populations, but the difference is not significant. At the upper nodes of the plant, mature leaf length declines steadily with plant age, allowing for a regression of leaf length on node. On average, the plants from more homozygous populations showed higher variance about the regression (MSE) and lower R² values, suggesting that the decline in leaf length with plant age is less stable in plants from selfing populations than in plants from outcrossing populations. Fluctuating asymmetry (FA) was calculated for four traits within single leaves at up to five nodes per plant. At the early nodes of the plant where leaf arrangement is opposite, FA was also calculated for the same traits between opposite leaves at a node. Fluctuating asymmetry is significantly greater in the southern selfing population than in the neighboring outcrossed population. Northern populations do not differ in FA. Fluctuating asymmetry can vary significantly between nodes. The FA values of different leaf traits were not correlated. We show that developmental stability can be measured in plants using FA and within-plant variance. Our data suggest that large differences in breeding system are associated with differences in stability, with more inbred populations being the least stable.     

Nucleophilic Distribution of Metallothionein in Human Tumor Cells

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 μMCdCl₂did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.     

Extracellular Matrix Penetration by Epithelial Cells Is Influenced by Quantitative Changes in Basement Membrane Components and Growth Factors

We have previously shown that isolated mouse fetal choroid plexus epithelial (CPE) cells penetrate a basement membrane matrix (Matrigel) substratein vitroto form single-layered epithelial vesicles embedded within the matrix. To determine which properties of the matrix are important for inducing or permitting cells to penetrate the substrate and organize into multicellular vesicles we have made quantitative changes to the basement membrane components and growth factors in cell cultures. Matrigel diluted to 33 or 10% with a collagen I gel was not permissive to cell invasion, and CPE cells formed a polarized epithelial monolayer on the substrate surface which had ultrastructural characteristics similar to those of CPE vesicles. Cells in these monolayers proliferated more rapidly than cells in epithelial vesicles. When deliberately embedded within a 33 or 10% Matrigel matrix, CPE cells were able to form vesicles, indicating that a dilute matrix is nonpermissive to cell invasion but promotes epithelial polarization and organization into vesicles. Cells embedded within a 100% collagen I matrix did not proliferate or form epithelial vesicles and the majority of cells did not remain viable. Addition of laminin to the collagen I gel promoted cell adhesion and cell survival, but did not promote the formation of extensive monolayers on the substrate nor the formation of epithelial vesicles within the matrix. Cell invasion into the 33% Matrigel matrix was induced by addition of laminin, nidogen, or a laminin–nidogen complex to the substrate or by addition of TGFβ2 to the culture medium, but not TGFβ1 or PDGF. These studies show that CPE cells are sensitive to quantitative changes in matrix composition, which influences their survival and proliferation and also their ability to penetrate the matrix and organize into multicellular epithelial vesicles.     

Interactions between the regulatory regions of twoAdh alleles

A region (NS1) that acts like an enhancer is located approximately 300 bp upstream of the larval cap site in theAdh gene ofD. melanogaster. When this sequence is deleted (NS1), the gene fails to express ADH protein. Gene expression can be restored by placing a secondAdh gene with an intact enhancer elsewhere on the same plasmid. In these circumstances, both genes are expressed equally regardless of their orientation on the plasmid. In this report we further characterize the interactions that occur when a single enhancer activates expression from a proximal and distant promoter. We have made the following observations: (1) While the two genes are expressed equivalently, their expression relative to a plasmid carrying two intact genes is reduced by a factor of 2 to 6 depending on the orientation of the two genes. (2) The single enhancer drives expression of both genes on any given plasmid molecule. (3) The enhancer does not interact with theAdh gene from which the NS7 region (which spans the larval TATA box) is removed. (4) Expression of the NS1 gene can be restored by an intact gene when both are inserted together into theDrosophila genome via P element-mediated transformation. (5) Increasing the separation between the two genes on a plasmid by up to 15 kbp does not prevent the restoration of expression of the NS1 gene. We propose a model that explains how a single enhancer can stimulate equal expression from two genes.     

Optimum growth conditions and light utilization efficiency of Spirulina platensis (= Arthrospira fusiformis) (Cyanophyta) from Lake Chitu, Ethiopia

Spirulina platensis (= Arthrospira fusiformis) was isolated from Lake Chitu, a saline, alkaline lake in Ethiopia, where it forms an almost unialgal population. Optimum growth conditions were studied in a turbidostat. Cultures grown in modified Zarrouk's medium and exposed to a range of light intensities (20–500 µmol photons m^–2s^–1) showed a maximum specific growth rate (µ_max) of 1.78 d^–1. Quantum yield for growth (µ) was 3.8% at the optimum light for growth of 330 µmol photons m^–2s^–1, and ranged from 2.8 to 9.4%. With increase in irradiance, the chlorophyll a concentration decreased, and the carotenoids/chlorophyll a ratio increased by a factor of 2.4. The phosphorus to carbon ratio (P/C) showed some variation, while the nitrogen to carbon ratio (N/C) remained relatively constant, thus causing fluctuations in the N:P ratio (7–11) of cells. An optimum N:P ratio of about 7 was attained in cells growing at the optimum light for growth. Results from the continuous culture experiments agreed well with maximum values of photosynthetic efficiency given in the literature for natural populations of S. platensis in the soda lakes of East Africa, Lake Arenguade (Ethiopia), and Lake Simbi (Kenya).     

Distribution and ecology of freshwater sponges in Connecticut

A survey of Connecticut lakes and rivers revealed the presence of 7 species of freshwater sponge: Spongilla lacustris, Ephydatia muelleri, Eunapius fragilis, Anheteromeyenia ryderi, A. argyrosperma, Corvomeyenia carolinensis, and Corvospongilla novaeterrae in order of decreasing frequency of occurrence. Corvomeyenia carolinensis has not been reported previously beyond its type locality in South Carolina. In addition, microscleres of Spongilla lacustris, Anheteromeyenia-like megascleres, Ephydatia muelleri-like megascleres, and smooth megascleres (amphioxeas), which could not be assigned to a particular species, were found in surface sediments from lake cores. Spongilla lacustris inhabiting small rivers produced brown, thick-capsuled gemmules during the summer and yellow, thin-capsuled gemmules during the fall. The thick-capsuled gemmules, but not the thin-capsuled gemmules, are tolerant of desiccation; and populations of Spongilla lacustris and Ephydatia muelleri survived severe drying of their habitats during the summer.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae

During periods of water deficit, plants accumulate late embryogenesis-abundant (LEA) proteins which are thought to protect cells from stresses associated with dehydration. One of these genes, le25, is expressed in tomato leaves and roots in response to water deficit and abscisic acid accumulation. To study the function of this protein and to test the effect of overproduction of the LE25 protein in Saccharomyces cerevisiae (Sc), a recombinant plasmid in which le25 is expressed under the control of the GAL1 promoter was constructed. The content of LE25 was high in Sc cells transformed with the recombinant plasmid. The transformant exhibited several stress-tolerant phenotypes. Growth of the transformant in a medium with 1.2 M NaCl was improved, as compared to a control strain. While the control strain showed a long lag phase of 40 h, le25-expressing cells showed a shortened lag phase of 10 h. However, no growth improvement was observed in a medium with 2 M sorbitol. In addition, the transformant had an increased survival rate after freezing stress, but not after high-temperature stress. These results, together with its predicted secondary structure, may indicate that LE25 functions as an ion scavenger.     

Mechanical effects of ET-1 in cardiomyocytes isolated from normal and heart-failed rabbits

Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca²⁺ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca²⁺ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca²⁺ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC₅₀ obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca²⁺ channels were similar with a peak amplitude at 1 nM ET-1 of –3.26 ± 0.8 in control cardiomyocytes and –3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca²⁺ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.Recipient of Servier Investigator Award