The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.     

Preparation of cytoplasmic bodies (cytospheres) from isolated hepatocytes and their biochemical properties

The controlled centrifugation of isolated rat hepatocytes at 260 000 g results in the formation of membrane-bounded cell fragments that we have termed 'cytospheres'. A method is described for the isolation of these cytospheres. Cytospheres are spherical, have a mean diameter of 9.2 +/- 3.2 microns (SD) and a protein content of 225 +/- 12 mg/g wet wt. About 3% of the protein from the original isolated hepatocyte suspension is recoverable. Transmission electron microscopy (TEM) shows cytospheres to possess a trilaminar membrane, and a finely granular hyaloplasm generally devoid of organelles, filaments and microtubules. Freeze-fracture studies reveal a membrane structure typical of a plasma membrane. Ouabain and wheat germ agglutinin (WGA)-binding studies indicate that the original orientation of the plasma membrane is maintained throughout the formation of the cytospheres. The cytospheres have also been characterized biochemically. Cytospheres are enriched in the enzymes normally associated with the hyaloplasm, whereas the activities of enzymes localized in organelles are greatly diminished. Lipid analysis of the cytosphere membrane indicates that it is derived from the plasma membrane of the hepatocyte. Cytospheres are sensitive to changes in the osmolarity and ionic composition of their environment. Cytospheres should therefore prove a useful preparation for the study of hyaloplasm metabolism and of plasma membrane receptor and permeability properties.     

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methodology for Estimating Numbers of Free-Living and Attached Bacteria in Estuarine Water

A fundamental problem in estuarine microbiology studies is the accurate determination of the density in the water column of both free-living bacteria and those attached to suspended particulate matter. When a water sample is filtered and the filter is viewed by epifluorescence microscopy, counts can be made of the numbers of bacteria which are seen on the filter background (free-living) and those which appear to lie on sediment particles (both free-living and attached). With only the additional knowledge of the proportion of the filter area covered by particles (a quantity that is straightforwardly determined by stereological point counting), results from geometric probability were used to determine the expected number of bacteria which are hidden by particles and hence to provide an estimation scheme for the true densities of free-living and attached bacteria. Variance equations based on a Taylor series are given, and a partial check of the method is attempted with controlled mixtures of bacteria and sediment. An alternative procedure is also proposed, in which the natural attached/free-living ratio is altered by an intervention experiment, allowing an estimation which is less model dependent but more labor intensive. Both methods are applied to a series of samples from the Tamar estuary, United Kingdom, taken in April 1985. A notable conclusion is that there are always more free-living than attached bacteria in the water column throughout the estuary.     

Distribution of Hydrogen-Metabolizing Bacteria in Alfalfa Field Soil

H₂ evolved by alfalfa root nodules during the process of N₂ fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H₂. Isolates showing net H₂ uptake and ³H₂ incorporation activity under laboratory conditions were assigned a Hup⁺ phenotype, whereas organisms with significant H₂ output capacity were designated as a Hout⁺ phenotype. Under aerobic isolation conditions two Hup⁺ isolates were obtained, whereas under microaerophilic conditions five Hup⁺ and two Hout⁺ isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H₂, and 1.2% could produce H₂. Two of the Hup⁺ isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C₂H₂ under the assay conditions suggested that the H₂ metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H₂ evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.     

Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Factors contributing to the seasonal variation of Bacillus spp. in pasteurized dairy products

The seasonal variation in the spoilage of pasteurized products, especially double cream, by spore-forming bacteria was due to a number of factors. By far the most important was the seasonal variation in the types of organisms isolated from raw milks. Psychrotrophic spore-formers predominated in the summer-autumn months and these strains were able to germinate rapidly and grow in refrigerated dairy products. There was evidence that the concentration of one or more factors which promoted germination of psychrotrophic strains of Bacillus spp. in milk was higher during the summer than in the winter. This again may contribute to seasonal differences in spoilage by spore-forming bacteria. Post-heat treatment contamination by spores of Bacillus spp. may also be more prevalent in the summer-autumn period and evidence was obtained that spores associated with post-pasteurization contamination could germinate and grow more rapidly than those introduced into the product from the raw material. Thus, the increased spoilage of pasteurized products by Bacillus spp. observed in the June to October period may be due to a combination of factors. The relative contribution that each makes is not easily resolved.     

Chemical limnology of two artificial lakes used for both stormwater management and recreation

The aims of this study were to document the mainly chemical behaviour of two linked artificial lakes used for both stormwater management and recreation in the new town of Craigavon. Further, the understanding of their behaviour should help in their management and the design of other similar lakes.The lake mean total phosphorus (73 µg P l^–1), nitrate (0.50 mg N l^–1) and chlorophyll a (25 µg l^–1) concentrations, Secchi depth (1.2 m) and the estimated total phosphorus loading (1.98 g m^–2 a^–1) all classify the main lake as eutrophic. An important source of the phosphorus load on the lakes is the urban area of Craigavon (52% of the total load). The interrelationships between total phosphorus, chlorophyll a and Secchi depth in the main lake are similar to those in natural ones. In addition, the lake follows the total phosphorus load — trophic state relationships (lake total phosphorus and chlorophyll a concentrations and Secchi depth) found to apply elsewhere. These two points indicate that the artificial lakes in Craigavon behave similarly to natural ones.     

Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase

Activation of rat liver phenylalanine hydroxylase by limited proteolysis catalyzed by chymotrypsin was investigated with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure gel filtration. Both activation and proteolysis were decreased by the addition of the natural cofactor, (6R)-tetrahydrobiopterin. From chymotryptic digests of the hydroxylase carried out in the presence and absence of (6R)-tetrahydrobiopterin, several different enzyme species were isolated by high pressure gel filtration. One species (subunit Mr = 47,000) with unchanged hydroxylase activity was isolated from the chymotryptic digest in the presence of (6R)-tetrahydrobiopterin; it was derived from the native enzyme (Mr = 52,000) by cleavage of the COOH-terminal Mr = 5,000 portion of the native enzyme. In the absence of (6R)-tetrahydrobiopterin, another species (subunit Mr = 36,000) was isolated. In addition to modification at the COOH-terminal end of the molecule, this species also had lost a Mr = 11,000 fragment from the NH2-terminal end of the hydroxylase. The Mr = 11,000 fragment was shown to include the phosphorylation site of the enzyme. This Mr = 36,000 species was 30-fold more active than the native phenylalanine hydroxylase when assayed in the presence of tetrahydrobiopterin. These results suggest that the regulatory domain that inhibits hydroxylase activity in the basal state may be located at the NH2 terminus of the phenylalanine hydroxylase subunit.