Delaying virus maturation for a higher yield of cell associated virus antigens

Negligible amounts of virus were released when infected cells were maintained in low ionic strength medium. The cells accumulated a high titre of virus and its specific antigenic proteins, detected by complement-fixation test. The procedure has potential for production of virus subunit vaccines.     

Preliminary studies on some coliphages from Kuwait

Seasonal data for raw sewage indicated a higher concentration of bacteriophages in the warmer months compared with the cooler months. Regardless of the amount of phages in the raw sewage and the time of year the efficiency of removal was as high as 83-96% for effluent II and 93-98% for sludge II. Four major types of plaque sizes were identified as Q1, Q2, Q3 and Q4. Although all isolates contained DNA, they showed variation in host range and heat sensitivity from 50 degrees C to 80 degrees C.     

Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative.

N-tert-butyloxycarbonyl (t-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-O-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli J5 to yield t-Boc-Cap-MLA. After a column purification step, the t-Boc group was removed by incubating the sample at low temperature in the presence of acid to yield Cap-MLA. This product was analyzed by californium plasma desorption mass spectrometry (PDMS). Purified t-Boc-Cap-MLA was further fractionated by reverse-phase high-performance liquid chromatography as its methyl ester and characterized by laser desorption mass spectrometry, PDMS, and proton nuclear magnetic resonance spectroscopy. These analyses revealed that the Cap group was selectively introduced into the 6'-position of MLA. To demonstrate that Cap-MLA can be conjugated to other compounds, it was reacted with biotin-Cap N-hydroxysuccinimide ester to yield biotin-(Cap)2-MLA. Analysis of this product by PDMS confirmed its expected molecular weight of 2171 and showed the presence of fragments containing the biotin and Cap groups. Monoclonal antibodies and streptavidin were used to show the presence of both lipid A and biotin in this conjugated product. These two novel lipid A derivatives were then tested for their bioactivities. Although both Cap-MLA and biotin-(Cap)2-MLA showed mitogenic activity using murine splenocytes, they were about 4-8 times less active than MLA at 20 micrograms/mL or less and only one-half as active at 100 micrograms/mL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diphosphoryl lipid A from Rhodobacter sphaeroides blocks the binding and internalization of lipopolysaccharide in RAW 264.7 cells.

Diphosphoryl lipid A derived from the nontoxic LPS of Rhodobacter sphaeroides (RsDPLA) has been shown to be a powerful LPS antagonist in both human and murine cell lines. In addition, RsDPLA also can protect mice against the lethal effects of toxic LPS. In this study, we complexed both the deep rough LPS from Escherichia coli D31 m4 (ReLPS) and RsDPLA with 5- and 30-nm colloidal gold and compared their binding to the RAW 264.7 cell line by electron microscopy. Both ReLPS and RsDPLA bound to the cells with the following observations. First, binding studies revealed that pretreatment with RsDPLA completely blocked the binding and thus internalization of ReLPS-gold conjugates to these cells at both 37 degrees C and 4 degrees C. Second, ReLPS was internalized via micropinocytosis (noncoated plasma membrane invaginations) involving formation of caveolae-like structures and leading to the formation of micropinocytotic vesicles, macropinocytosis (or phagocytosis), formation of clathrin-coated pits (receptor mediated), and penetration through plasma membrane into cytoplasm. Third, in contrast, RsDPLA was internalized predominantly via macropinocytosis. These studies show for the first time that RsDPLA blocks the binding and thus internalization of LPS as observed by scanning and transmission electron microscopy.     

Lipid A-like molecules that antagonize the effects of endotoxins on human monocytes

Lipopolysaccharide (LPS) endotoxin is implicated as the bacterial product responsible for the clinical syndrome of Gram-negative septicemia. Although the lipid A domain of LPS appears to be responsible for the toxicity of endotoxin, lipid A from the photosynthetic bacterium Rhodobacter sphaeroides (RSLA) and a disaccharide precursor of lipid A from enteric bacteria, termed lipid IVA, have little activity on human cells. Using the human promonomyelocytic cell line THP-1 and human monocytic cells, we now show that both lipid IVA and RSLA are antagonists of LPS. Complete, apparently competitive, inhibition of LPS activity is possible at a 10-100-fold excess of antagonist, as judged by measuring the release of cytokines and prostaglandin E2. Both antagonists prevent monocyte stimulation by endotoxin extracted from a variety of Gram-negative bacteria. Cells pretreated with either inhibitor and subsequently washed still show attenuated responses to LPS. Stimulation of monocytes by whole Gram-negative bacteria is also antagonized in a dose-dependent manner. Lipid X has no inhibitory effect in the same dose range as lipid IVA and RSLA. These findings rule out LPS sequestration as the explanation for the observed antagonism. Neither inhibitor alters monocyte stimulation by phorbol 12-myristate 13-acetate, Staphylococcus aureus, or purified protein derivative, demonstrating specificity for LPS. Although RSLA appears to inhibit LPS when tested with macrophages from both humans and mice, lipid IVA had the unique ability to act as an LPS antagonist with human-derived cells but to exhibit LPS-like effects with murine-derived cells. Like LPS, lipid IVA stimulated the release of both tumor necrosis factor alpha and arachidonic acid from murine-derived RAW 264.7 macrophage tumor cells. The range of concentrations necessary for lipid IVA to induce LPS-like effects in murine cells was similar to that necessary to antagonize the actions of LPS in human monocytes. The agonist activities of lipid IVA were completely inhibitable by RSLA. This unique species-dependent pharmacology observed with lipid IVA may reflect differences between human and murine LPS receptors. RSLA and lipid IVA may be useful in defining the role of LPS in Gram-negative bacterial infections and may prove to be prototypical therapeutic agents for the treatment of Gram-negative septicemia.     

Structure of a Micrococcus lysodeikticus cell wall fragment containing phosphorylated sugars.

A polysaccharide-peptidoglycan complex containing different phosphorylated sugars from Micrococcus lysodeikticus cell wall has been isolated and purified. The peptidoglycan contained muramic acid 6-phosphate and N-acetylglucosamine 6-phosphate as phosphorylated sugars in addition to other sugar residues. Mild acid hydrolysis of the peptidoglycan and subsequent reduction of the released polysaccharide showed therein the presence of glucose and N-acetyl-glucosamine in the linkage of the external polysaccharide residues to the peptidoglycan through phosphodiester linkage. These data suggest the presence of polysaccharide chains linked to a peptidoglycan core through two phosphorylated sugars via two different terminal carbohydrate residues of the external polysaccharide chains in a same polymer.     

Antidiuretic hormone infusion reduces taurine and NaCl-induced hypernatremia in the rats

Summary Rats drinking taurine and hypertonic saline (T + S) develop severe hypernatremia, but rats drinking either T or S alone do not. One hypothesis for this disruption of homeostasis is that the T + S combination interferes with the actions of antidiuretic hormone (ADH). Rats drinking T + S developed severe hypernatremia (170 mmol/L) by day 8 when infused with distilled water by osmotic minipumps, but maintained plasma sodium below 150 mmol/ L when infused with ADH. Cumulative water balance in T + S drinkers receiving ADH was consistently higher than in those not receiving ADH. However the ratio of cumulative sodium balance to cumulative water balance suggests little uniform advantage to rats receiving ADH nor does comparison of urine osmolality in the two groups. Precisely how ADH administration reduces hypernatremia in T + S drinking rats remains unclear, but the hypothesis that T + S interferes with the action of ADH in its regulation of extracellular fluid volume and osmolality remains viable.Supported by FMHS Project Grant CP/96/22 and St. George's University.