Loe de Leij

An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli.

The inner and outer membranes of gram-negative bacteria are usually obtained from spheroplasts. The lysozyme treatments normally used for converting cells to spheroplasts were originally developed with exponential phase cells and have proven to be ineffective with cells grown under other conditions. A procedure has therefore been developed which renders variously grown cells completely susceptible to lysozyme. This procedure has been tested on various strains of Escherichia coli at all stages of growth in minimal medium, from the early exponential to the late stationary phase. It has been tested on stationary phase cultures which ceased to grow because of limiting aeration, limiting carbon source, limiting amino acids, and limiting nicotinic acid. Very efficient conversion of cells to spheroplasts was observed in all cases. The resulting spheroplasts are an excellent source for subsequent membrane separations.

Release of outer membrane fragments from normally growing Escherichia coli

A complex containing lipopolysaccharides, phospholipids and proteine separated from the medium by gelfiltration on Sephadex G-200 or by centrifugation. Electron microscopy revealed that this material is released as vesicles and membrane fragements. To determine the origin of these fragments, they were compared to outer and cytoplasmic membranes with respect to keto-deoxyoctulosonic acid, phospholipid, and protein content, phospholipid composition, fatty acid composition, protein distribution on sodium dodecyl sulfate-polyacrylamide gels, buoyant density, and content of several membrane marker enzymes. The results of this comparison indicate that the membrane fragments found in the culture supernatant of normally growing Escherichia coli consist of practically unmodified outer membrane. Possible mechanisms as to the cause of the release of outer membrane fragments, and its relationship to cell-division, are discussed.

The immune response during the luteal phase of the ovarian cycle : a Th2-type response?

OBJECTIVE To test the hypothesis that during the luteal phase of the ovarian cycle, as compared with the follicular phase, the peripheral immune response is shifted toward a type-2 response. DESIGN Prospective study. SETTING Academic research setting. PATIENT(S) Women with regular menstrual cycles. INTERVENTION(S) Blood samples were collected between days 6 and 9 of the menstrual cycle and 6-9 days after the LH surge. MAIN OUTCOME MEASURE(S) Intracellular cytokine production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10 after in vitro stimulation of lymphocytes as well as total white blood cell (WBC) count, differential WBC count, and plasma 17 beta-E(2) and P concentrations. RESULT(S) Mean plasma 17 beta-E(2) and P concentrations, WBC count, and mean granulocyte, monocyte, and lymphocyte counts were significantly increased in the luteal phase as compared with the follicular phase of the ovarian cycle. Production of type-1 cytokines (IFN-gamma and IL-2) and production of the type-2 cytokine IL-10 did not vary between the phases of the ovarian cycle. Production of the type-2 cytokine IL-4, however, was significantly increased in the luteal phase as compared with the follicular phase of the ovarian cycle. CONCLUSION(S) During the luteal phase of the ovarian cycle, the immune response is shifted toward a Th2-type response, as reflected by increased IL-4 production in this phase of the cycle. These results may suggest that increased levels of P and 17 beta-E(2) in the luteal phase of the ovarian cycle play a role in the deviation of the immune response toward a type-2 response.

How does lysozyme penetrate through the bacterial outer membrane

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme. In the presence of Mg2+, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg2+ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg2+. Mg2+ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose. These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.

The immune response during the luteal phase of the ovarian cycle: increasing sensitivity of human monocytes to endotoxin

OBJECTIVE To test the hypothesis that during the luteal phase of the human ovarian cycle, as compared with the follicular phase, the percentage of cytokines producing peripheral monocytes after in vitro stimulation with endotoxin is increased. DESIGN Prospective study. SETTING Academic research institution. PATIENT(S) Women with regular menstrual cycles. INTERVENTION(S) Blood samples were collected between days 6 and 9 of the menstrual cycle (follicular phase) and between days 6 and 9 of the menstrual cycle following the LH surge (luteal phase). MAIN OUTCOME MEASURE(S) Percentages of tumor necrosis factor (TNF)-alpha-, interleukin (IL)-1 beta-, and IL-12-producing monocytes as well as total white blood cell (WBC) count, differential WBC counts, and plasma 17 beta-estradiol and progesterone concentrations. RESULT(S) Mean plasma 17 beta-estradiol and progesterone concentrations, percentage of TNF-alpha- and IL-1 beta-producing monocytes, WBC counts, and granulocyte cell count were significantly increased in the luteal phase as compared with the follicular phase of the ovarian cycle. The percentage of IL-12-producing monocytes, monocyte count and lymphocyte count did not vary between the 2 phases of the ovarian cycle. CONCLUSION(S) Together with an increase in progesterone and 17 beta-estradiol during the luteal phase, there is an increase in percentage TNF-alpha- and IL-1 beta-producing peripheral monocytes after in vitro stimulation with endotoxin as compared with the follicular phase of the ovarian cycle. Whether this increased sensitivity of monocytes for proinflammatory stimuli during the luteal phase is due to increased plasma levels of progesterone or 17 beta-estradiol needs further investigation.

Structural heterogeneity of the cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes of gram-negative bacteria can be isolated from spheroplasts, and separated on sucrose density gradients. Lysis of spheroplasts causes extensive membrane fragmentation and since the characteristics of the fragments obtained by different lysis procedures need not be identical, the influence of the disruption method on membrane composition has been examined. Spheroplasts of Escherichia coli J5 were lysed by osmotic shock, which did not significantly separate the cytoplasmic and outer membranes, but resulted in mixed membrane vesicles. Lysis in the French press and by sonication caused extensive membrane fragmentation and separation. Sonication, however, also caused some fusion between fragments of the outer and the cytoplasmic membranes; this intermembrane fusion increased with sonication time. When the cytoplasmic and outer membranes were well separated and intermembrane fusion was minimal or absent, the cytoplasmic and outer membrane fragments were heterogeneous with respect to density and ovarll phospholipid, protein and lipopolysaccharide composition. In addition, cytoplasmic, but not outer, membrane fragments were also heterogeneous with respect to protein composition. It is concluded, therefore, that membrane fragments obtained from the cytoplasmic and outer membranes are heterogeneous independently of the lysis procedures used to obtain these fragments. Possible reasons for this heterogeneity are discussed.

Diagnostic application of a monoclonal antibody against small cell lung cancer

A monoclonal antibody (MOC‐1) directed against an antigen present in small cell lung cancer (SCLC) was used for diagnostic purposes. After screening of biopsy specimens of lung tumors, MOC‐1 was found to react with SCLC (n = 10) and adenocarcinoma of the lung (4 of 9 cases). Except for a few cells in a poorly differentiated tumor, the reaction with squamous cell cancer was negative (n = 6). Staining with MOC‐1 by an immunoperoxidase technique on imprints of biopsy specimens procured by rigid bronchoscopy was found to be a reliable and rapid method for diagnosing SCLC (16 of 17 positive). All cytologically proven bone marrow and pleural metastases of SCLC were found by staining on a cytospin preparation with MOC‐1. Moreover, in three cytologically negative cases, MOC‐1‐positive cells were detected.

Detection and localization of peptidases in Lactococcus lactis with monoclonal antibodies

Monoclonal antibodies against peptidases of Lactococcus lactis were isolated and characterized: PEPN1-4 against a lysyl aminopeptidase PepN, PEPT1-5 against a tripeptidase PepT and PEPD1-3 against a dipeptidase PepD. These monoclonal antibodies reacted specifically with their respective antigens in crude cell extracts of Lc. lactis subspp. cremoris and lactis. A number of monoclonal antibodies cross reacted with proteins of other (lactic acid) bacteria. PEPT1, 2, 4 and 5 cross reacted weakly with a 35 kDa protein in Lactobacillus delbrueckii, while PEPT1 and PEPT2 reacted with proteins in the cell-free extract of Streptococcus thermophilus and Clostridium fervidus. Of the four isolated monoclonal antibodies against PEPN, only PEPN3 cross reacted weakly with a 90 kDa protein in Escherichia coli cell-free extract, and the other three antibody species against PEPN3 cross reacted with 80 kDa proteins of Lb. casei, Lb. delbrueckii, and Str. bovis, but not of Esch. coli. Of the three monoclonal antibodies against PepD, only PEPD1 and PEPD2 cross reacted with 40 kDa proteins of Lb. casei, Lb. delbrueckii and Str. bovis. All PEPN, PEPD and PEPT antibodies reacted with components in cell-free extracts of eleven different Lc. lactis strains, indicating that the peptidases of these strains were very similar to those of Lc. lactis subsp. cremoris WG2. However, Lc. lactis subsp. hordniae appeared to differ from the other Lc. lactis subspecies since only PEPT1, 2 and 5 reacted with a protein in the cell-free extract. Immunogold labelling of Lc. lactis WG2 with the isolated monoclonal antibodies revealed that PepN, PepD and PepT were located intracellularly. The intracellular location of these peptidases is discussed in relation to the supply of essential amino acids and peptides.