Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.

While searching for a phospholipase C (PLC) specific for phosphatidylcholine in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold. The secreted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol. The active enzyme is a monomer of approximately 40 kDa. As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for activity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2. The compound D609, described to selectively inhibit phosphatidylcholine-specific PLCs, caused half-inhibition of the P. fluorescens enzyme at approximately 420 microM, while 50-fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens. Partial peptide sequences obtained from the pure P. fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P. fluorescens gene library prepared from our laboratory isolate. It contains an ORF of 1155 nucleotides encoding the PLC. There is no significant sequence homology to other PLCs, suggesting that the P. fluorescens enzyme represents a distinct subclass of bacterial PLCs. The protein lacks cysteine residues and consequently contains no disulfide bonds. Interestingly, P. fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant coding sequence.

Two different functions for CD44 proteins in human myelopoiesis.

CD44 is important during myelopoiesis, although the contributions of variant CD44 proteins are unclear. We show here that in human long-term bone marrow culture antibodies recognizing a CD44 NH2-terminal epitope (mab 25-32) or a CD44v6 epitope (mab VFF18) inhibit myelopoiesis. However, mab 25-32 but not mab VFF18 affects myeloid colony formation. These data suggest that an early precursor cell compartment is the target for the 25-32 antibody, whereas the mab VFF18 targets later stages in myelopoiesis. Since the bulk of hemopoietic precursor cells are negative for the v6 epitope and only a minor subset of myeloid cells express the v6 epitope, we have used several human myeloid progenitor cell lines to unravel the function of different CD44 proteins. These cell lines produce variant CD44 proteins, predominantly a new variant CD44v4-v10, when stimulated towards myeloid differentiation. Features that can be acquired by the expression of CD44v4-v10 are an increased hyaluronate (HA) and a de novo chondroitin sulphate A (CS-A) binding. Although, the expression of CD44v4-v10 per se is necessary for HA and CS-A binding, the protein backbone seems to require appropriate glycosylation. HA binding results in CD44-mediated cellular self-aggregation and adhesion to the stromal cell line MS-5. In summary, our data suggest that different CD44 proteins are important for at least two different steps in myelopoiesis.

Protective effect of O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxic and recombinogenic activity of different antineoplastic drugs.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine in DNA and thus may protect cells against genotoxic effects of agents inducing this lesion. To analyze quantitatively the level of protection mediated by MGMT against antineoplastic drugs, we determined the cytotoxic and recombinogenic (sister-chromatid exchange inducing) effects of various chemotherapeutic agents in a pair of isogenic Chinese hamster cell lines deficient and proficient for MGMT, generated upon transfection with human MGMT cDNA. Furthermore, we compared the responses of the human cell lines HeLa MR (MGMT deficient) and HeLa S3 (MGMT proficient) to the various agents. It is shown that: (1) MGMT proficient cells are resistant in cell killing to the methylating drug streptozotocin and all the chloroethylating nitrosoureas tested. There was a marked agent specificity in protection. The level of resistance provoked by MGMT increased in the order BCNU < CCNU < ACNU < HeCNU < streptozotocin. (2) MGMT did not protect cells against killing induced by chlorambucil, cisplatin, melphalan, activated cyclophosphamide (malosfamide) and activated ifosfamide (4-hydroperoxy-ifosfamide). (3) MGMT caused protection against the recombinogenic effect of all nitrosoureas tested. The lowest level of protection was again observed for BCNU, followed by CCNU, ACNU < HeCNU < streptozotocin. (4) MGMT proficient cells did not exhibit resistance in SCE induction towards cyclophosphamide (activated by microsomes), 4-hydroperoxy-ifosfamide, mafosfamide, chlorambucil and melphalan. Some protection was afforded, however, against cisplatin (and transplatin). This effect was abolished by pretreatment of cells with O6-benzylguanine, which depletes MGMT, indicating that some lesion(s) induced by cisplatin giving rise to SCEs can be repaired by MGMT. Taken together, these results indicate that streptozotocin, HeCNU and ACNU are more selective than CCNU and BCNU in killing MGMT deficient cells, and that in the cases of cyclophosphamide, ifosfamide, chlorambucil, cisplatin and melphalan MGMT is not involved in mediating cytotoxic drug resistance.

Targeting of O6-methylguanine-DNA methyltransferase (MGMT) activity by antimessenger oligonucleotide sensitizes CHO/Mex+ transfected cells to mitozolomide.

The targeting of mRNA with antisense oligonucleotides is increasingly employed to inhibit the expression of gene function. Since the level of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is decisive in protection of cells against damage produced by alkylating agents, including cytostatic drugs, the targeted inhibition of this repair activity might be of importance for therapeutic approaches. In order to investigate whether antisense targeted MGMT depletion is feasible to transiently modify the sensitivity of cells to anticancer drugs, we studied the expression of MGMT and cellular sensitivity upon inhibitor and antisense treatment using CHO transfectants expressing human MGMT. It was shown by polymerase chain reaction that antisense oligonucleotides specifically inhibited MGMT mRNA level. Nevertheless, MGMT protein was found not to be reduced significantly, as demonstrated by Western blotting. Correspondingly, no significant decrease in MGMT activity was observed, as measured 36 h after MGMT antisense oligonucleotide administration. Given together with the MGMT depleting agent O6-methylguanine, reduction in MGMT protein as well as activity was found. MGMT antisense oligonucleotide enhanced the sensitivity of cells to the tumor therapeutic drug mitozolomide, as measured by sister chromatid exchange formation. This sensitization was further enhanced by combined treatment with antisense oligonucleotide and O6-methylguanine, indicating that MGMT antisense can be supportive in sensitization of cells to an alkylating drug.

Activity of the DNA repair protein O6-methylguanine-DNA methyltransferase in human tumor and corresponding normal tissue.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a main determinant of resistance of cells towards the cytostatic effect of N-nitrosoureas. Here we report the activity of MGMT in different types of human tumors. Colorectal tumors showed the highest mean of MGMT activity (481 +/- 258 fmol/mg protein) followed by ovarian tumors (437 +/- 291 fmol/mg), breast (306 +/- 204 fmol/mg), testicular (299 +/- 179 fmol/mg), and brain tumors (55 +/- 44 fmol/mg). Only in breast and brain tumors were MGMT-deficient samples found, with a frequency of 3 and 5%, respectively. No significant difference in mean MGMT activity was observed between breast tumors of grading I, II, and III. For brain tumors there was a tendency of MGMT to decline with increasing grading. Breast and colorectal carcinomas were compared with the corresponding normal tissue obtained from the same patient. The data showed for 11 out of 12 pairs of breast tissue and for 13 out of 15 pairs of colorectal tissue that MGMT activity in the tumor was equal to or, more frequently, higher than the activity found in the corresponding normal tissue. Thus, it appears that in these groups of tumors, increase of MGMT activity during tumor formation and progression is the rule rather than the exception.

A novel phospholipase A2 from human placenta.

A major soluble phospholipase A2 of human term placenta was characterized and purified about 15,000-fold to homogeneity. The apparent molecular mass as determined in SDS/polyacrylamide gels is 42 kDa. The enzyme is inhibited by dithiothreitol indicating the presence of disulphide bridges which are essential for activity. Studies with known phospholipase A2 inhibitors revealed no immediate relationship to either secretory or cytosolic phospholipases A2. The placental enzyme prefers liposomes of phosphatidylcholine and has a distinct preference for arachidonic acid in the sn-2 position. It tolerates various detergents. Roughly 10 microM Ca2+ is required for activity, but it cannot be replaced by Mg2+ or Mn2+; Zn2+, Cu2+ and Fe3+ are inhibitory. In immunoblots, the placental enzyme was not detected by two separate antisera specific for type-II phospholipases A2 but reacted very weakly with antisera directed against cytosolic phospholipase A2. From these data we suggest that this enzyme is a novel form of phospholipase A2 which may be involved in arachidonic acid mobilization both during the course of pregnancy and at parturition.

O6-methylguanine-DNA methyltransferase activity in breast and brain tumors.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a main determinant of resistance of tumor cells to the cytostatic activity of chemotherapeutic alkylating agents (methylating and chloroethylating nitrosoureas) and is effective in protecting normal cells against genotoxic and carcinogenic effects resulting from DNA alkylation. Therefore, the level of expression of MGMT is significance for the response of both the tumor and the non-target tissue following application of nitrosoureas in tumor therapy. To determine the expression of MGMT in tumor tissue, we have assayed MGMT activity in 68 breast carcinomas and 38 brain tumors. There was a wide variation of MGMT expression in breast carcinomas ranging from below the level of detection up to 863 fmol/mg protein. About 4% of breast tumors did not display detectable MGMT, 15% had activity lower than 100 fmol/mg protein, and 26% expressed more than 500 fmol/mg. The mean level of expression was 321 fmol/mg. In brain tumors (astrocytoma WHO grade I, II, and III, and glioblastoma WHO grade IV) the MGMT activity was generally lower than in breast tumors, ranging from below the level of detection up to 238 fmol/mg. The mean level of expression was 55 fmol/mg. Five percent of the brain tumors had no detectable MGMT activity. The MGMT repair activity correlated well with the amount of MGMT protein present in tumor samples, as shown by Western-blot analysis, indicating that loss of MGMT repair activity is due to inability of these tumor cells to synthesize the protein.

Regulation of the concentration of free arachidonic acid in homogenates of human platelets.

With the intention to study the regulation of the availability of free arachidonic acid through the enzymes of the Lands cycle, we established a model system in homogenates of human platelets. Phospholipase A2, arachidonoyl-CoA synthetase and lysophosphatidyl acyltransferase proved to be simultaneously active and a steady turnover of arachidonic acid was the consequence. EGTA suppressed the deacylating activity that acted on endogenous membrane phospholipids and prevented eicosanoid formation from previously esterified exogenous arachidonoyl-CoA. The reacylating enzymes took part in the control of eicosanoid biosynthesis by re-esterification of liberated arachidonic acid. Blockade of the reacylation by apyrase made arachidonic acid completely available for further metabolization into 12-HETE and thereby induced an increase in the eicosanoid release.

[Activity in vitro and pharmacokinetics of minocycline (author's transl)]. / In-vitro-Aktivität und Pharmakokinetik von Minocyclin

The in vitro activity of doxycycline and minocycline (Klinomycin) was determined by serial dilution test in 100 strains of E. coli, 101 strains of enterobacter, 91 tetracycline-sensitive and 52 tetracycline-resistant strains of staphylococci. Only staphylococci were more sensitive against minocycline than against doxycycline whereas other species showed nearly the same sensitivity against both antibiotics. After i.v. infusion of 200 mg minocycline (during 1 h) mean serum levels fell from 3.5 mug/ml to 0.6 mug/ml (after 24 h). Half-life was calculated as 15.7 h, urine recovery as 5.9%. After oral application of 200 mg minocycline serum level peaks were 2.7 mug/ml, serum levels after 24 h 0.7 mug/ml. At repeated administrations daily dosage of 100 mg was too low of 200 mg sufficient to obtain the same serum levels as after the initial dose of 200 mg. CSF levels after oral administration of 0.4 g minocycline were 0.74 +/- 0.09 mug/ml (in serum at the same time 2.2 +/- 0.2 mug/ml). Half-life of minocycline in chronic renal failure (7 adult patients) was not prolonged (15--20 h). Minocycline is especially suitable for treatment of infections of unknown bacterial origin (including such caused by staphylococci). I.v. infusion is indicated only in unconscious or vomiting patients.

[The pharmacokinetics of doxycycline in kidney insufficiency in geriatric patients compared to younger adults]. / Die Pharmakokinetik von Doxycyclin bei Niereninsuffizienz und geriatrischen Patienten im Vergleich zu jüngeren Erwachsenen

In young adults i.v. injection of 200 mg doxycycline was followed by a rapid fall in serum levels in the first 30 minutes and than a slow decrease (serum concentration after 24 h on average 1.0 mug/ml). Biological half-life was 11.9 h, final distribution volume 50.0 litres and urine recovery 70%. After oral administration of 200 mg doxycycline the serum peaks measured were 3.4 mug/ml (2 1/2 h later). With repeated doses of 100 mg every 24 h serum peaks were 2.8 mug/ml and serum concentrations after 24 h 0.8-0.9 mug/ml (urine recovery 43%). The area under the curve was 25.5% less than after i.v. injection. In 7 adults with chronic renal failure the half-life of doxycycline varied between 10 and 24 h. With repeated oral administration of doxycycline (100 mg every 24 h) there was no accumulation of the drug in blood. During hemodialysis (Stuttgart kidneys, Rhône-Poulenc plates) doxycycline injected i.v. was eliminated as rapidly as before. In renal failure doxycycline may be given in the same dosage as where renal function is normal. In 12 geriatric patients without renal disease, serum levels of doxycycline after i.v. injection of 200 mg were not significantly different from those of young adults (distribution volume 46.2 +/- 16.2 litres). It can therefore be assumed that tissue penetration of the drug is similar in the elderly and in young adults.