Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.     

The effect of endogenous essential and nonessential fatty acids on the uptake and subsequent agonist-induced release of arachidonate

We have demonstrated that the uptake and agonist-induced release of a pulse of arachidonate are influenced by the size and composition of preexisting endogenous fatty acid pools. EFD-1 cells, an essential fatty acid-deficient mouse fibrosarcoma cell line, were incubated with radiolabeled (14C or 3H] arachidonate, linoleate, eicosapentaenoate (EPA), palmitate, or oleate in concentrations of 0-33 microM for 24 h. After 24 h, the cells were pulsed with 0.67 microM radiolabeled (3H or 14C, opposite first label) arachidonate for 15 min and then stimulated with 10 microM bradykinin for 4 min. Because EFD-1 cells contain no endogenous essential fatty acids, we were able to create essential fatty acid-repleted cells for which the specific activity of the newly constructed endogenous essential fatty acid pool was known. Loading the endogenous pool with the essential fatty acids arachidonate, eicosapentaenoate, or linoleate (15-20 nmol of fatty acid incorporated/10(6) cells) decreased the uptake of a pulse of arachidonate from 200 to 100 pmol/10(6) cells but had no effect on palmitate uptake. The percent of arachidonate incorporated during the pulse which was released upon agonist stimulation increased 2-fold (4-8%) as the endogenous pool of essential fatty acids was increased from 0 to 15-20 nmol/10(6) cells. This 8% release was at least 3-fold greater than the percent release from the various endogenous essential fatty acid pools. In contrast, loading the endogenous pool with the nonessential fatty acids oleate or palmitate to more than 2-3 times their preexisting cellular level had no effect on the uptake of an arachidonate pulse. Like the essential fatty acids, increasing endogenous oleate increased (by 2-fold) the percent release of arachidonate incorporated during the pulse, whereas endogenous palmitate had no effect on subsequent agonist-induced release from this arachidonate pool. These studies show that preexisting pools of essential and nonessential fatty acids exert different effects on the uptake and subsequent releasability of a pulse of arachidonate.     

Preparation of rat monoclonal antibodies to epitopes encoded by the viral oncogene (v-fms) of McDonough feline sarcoma virus

The McDonough strain of feline sarcoma virus (SM-FeSV) contains a viral oncogene, v-fms, transduced from cat cellular genetic sequences designated c-fms. Monoclonal antibodies reactive to antigenic determinants encoded by v-fms were prepared by immunizing rats with live, syngeneic SM-FeSV-transformed cells, and fusing splenic lymphocytes from a tumor-bearing animal with cultured rat myeloma cells. Culture supernatants from hybrids producing antibodies to epitopes encoded by v-fms were identified by immunoprecipitation of radiolabeled polypeptides from SM-FeSV-transformed mink cells. Four positive hybrids were cloned twice in soft agar, established as stable lines, and grown in defined serum-free medium to facilitate purification of homogeneous antibodies. The monoclonal antibodies were used to assay SM-FeSV-specific products by "immunoblotting" of electrophoretically separated proteins, and by fixed-cell immunofluorescence.     

Characterization of the efficiency of a cell separation process by the extent of elimination of a contaminating cell type

A stepwise approach to the selection of an appropriate technique for a cell separation problem is presented in which the preparative purification of cells is linked to their analytical separation. We have introduced the extent of elimination of a contaminating cell type from the cell type which one chooses to purify, as a separation parameter that characterizes the efficiency of a separation process independently of the relative cell composition of the starting material. In order to compare different separation techniques, a preparative fraction boundary needs to be chosen between the cell types. We defined this boundary in terms of the physical property on which the separation is based such that yield and purity of the isolated cell suspension are optimized simultaneously. With this analytical approach, it was found that a similar elutriation technique separated human and equine mononuclear cells equally well and that the separability of human monocytes and lymphocytes improved when the cells were separated by increasing the limiting sedimentation coefficient value of the elutriation chamber in small increments.     

Ageing of the human corneal stroma: structural and biochemical changes.

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm2 to 3.46 nm2, and an increase in scleral collagen from approx. 2.65 nm2 to 3.19 nm2. These changes may be due to an increase in the extent of non-enzymic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

IFN-gamma-induced L-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis factor-alpha.

Activated murine peritoneal macrophages inhibit the intracellular proliferation of Toxoplasma gondii and produce a number of cytokines, such as TNF-alpha and IL-1. Both TNF-alpha and IL-1 have been reported to be involved in the immune response against various microorganisms, but the mechanisms responsible for these effects are not known. In the present study it was investigated whether endogenously produced TNF-alpha and IL-1 are involved in the activation of peritoneal macrophages by rIFN-gamma leading to toxoplasmastatic activity and the production of reactive nitrogen intermediates. The rIFN-gamma-induced toxoplasmastatic activity was inhibited by neutralizing antibodies against mouse TNF-alpha in a dose-dependent and time-dependent way, but neutralizing antibodies against mouse IL-1 alpha and IL-1 beta did not affect this activity. Involvement of TNF-alpha in the induction of toxoplasmastatic activity was confirmed by our finding that rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma inhibited the intracellular proliferation of T. gondii. No synergistic activity of rIL-1 and rIFN-gamma on the inhibition of T. gondii proliferation was found. Both rTNF-alpha and rIL-1 alpha alone inhibited the intracellular proliferation of T. gondii only slightly. Because it has been reported recently that activated macrophages produce reactive nitrogen intermediates that are essential in the induction of toxoplasmastatic activity, we investigated whether these intermediates are involved in the TNF-dependent induction of toxoplasmastatic activity. Neutralizing antibodies against mouse TNF-alpha inhibited also the release of NO2- by rIFN-gamma-activated macrophages almost completely. Macrophages incubated with rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma released substantial amounts of NO2-, but rTNF-alpha and rIL-1 alpha alone, and the combination of rIL-1 alpha and a nonactivating concentration of rIFN-gamma induced only little NO2(-)-release by macrophages. To assess whether reactive nitrogen intermediates act directly or indirectly on the intracellular proliferation of T. gondii, macrophages were incubated with the L-arginine analog NG-monomethyl-L-arginine or the NADPH-inhibitor diphenylene iodonium, both inhibitors of the generation of reactive nitrogen intermediates. Good correlation was found between toxoplasmastatic activity and the release of NO2- during the 24-h activation period before infection of the macrophages with T. gondii, but no correlation was found between toxoplasmastatic activity and the release of NO2- during infection of the macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human lactase and the molecular basis of lactase persistence

Human lactase purified from detergent extracts of the total membrane fraction of postmortem jejunum by means of monoclonal immunoadsorbent chromatography appears to be a dimer of subunits identical in M_r (160K). Trypsin or papain removes a small hydrophobic anchoring peptide from each subunit to give a hydrophilic enzyme which no longer interacts with detergent micelles. Lactase hydrolyzes, besides lactose, cellobiose and the synthetic substrates, 4-methylumbelliferyl--galactoside and -glucoside, as well as phlorizin; but it does not hydrolyze glucocerebroside. Phlorizin hydrolase is associated with lactase under all conditions investigated; coincident staining on immunodiffusion and immunoelectrophoresis, coincident elution on immunoadsorbent chromatography and on gel filtration in a dissociating buffer, and correlated reduction in activity in lactase-nonpersistent individuals. Adult and infant lactases are indistinguishable by titration or immunodiffusion against polyclonal rabbit antibodies. Adult individuals low in lactase activity also show a corresponding reduction in cross-reacting material. These observations suggest that lactase persistence is due to the continued synthesis of the infant enzyme.Financial support was provided by the Nuffield Foundation, the Medical Research Council, and the Open University Research Committee Fund.     

Cell surface characteristics and DNA content of macrophages in murine bone marrow cultures

Summary An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied immediately thereafter with the SEM part of the instrument. The cells were divided into six groups according to the number of microvilli and/or microridges present on their surface. A proportion of macrophages showed a DNA content more than occurs in diploid cells, which could indicate a future division. No special surface morphology could be detected in this cell type.     

Biochemical and evolutionary aspects of arthropod predation on ferns

Summary The widely held assumption that very few arthropods feed on ferns was questioned following field observations of arthropod damage on ferns in the state of Veracruz, Mexico. The extent and type of damage was recorded and it was found that in a measured locality, ferns were no less attacked than the angiospermous flora. As chemistry and arthropod host relationships have been shown to be so closely intertwined, plants collected in the field were analysed for both condensed tannins and cyanogenic glycosides, compounds known to be effective deterrents in temperate climates. Although all ferns tested contained tannins these did not appear to inhibit predation. Cyanogenic glycosides were present in only 3% of the fern species analysed, and it is, therefore, unlikely that they play a significant role as defensive compounds in the ferns examined.A literature search revealed a large number of ferns cited as being arthropod hosts. Approximately 420 named species of arthropods have been recorded, the majority of which are from the orders Coleoptera, Hymenoptera, Lepidoptera, and Hemiptera. Both evolutionary primitive (sawflies) and advanced (moths) arthropods are reported to be present on ferns suggesting possible coevolution of arthropods and ferns both before and after the radiation of angiosperms.     

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.