[Isolation and biotyping of Streptococcus mutans among preschool children in Kaohsiung city].

Strains of streptococcus mutans were isolated from the saliva of preschool children in Kaohsiung City. The unstimulated whole saliva samples were inoculated onto modified Gold's agar plates and incubated for 24-48 hours anaerobically at 37 degrees C. Strains of S. mutans were classified according to Shklair and Keene's biochemical methods and biotyped according to the methods of Farghaly et al. S. mutans could be detected in 102 (89.5%) of 114 preschool children. The most common biotype of S. mutans in single isolation was biotype I (72.9%). The percentages of the other biotypes are as follows: biotype II (5.2%), biotype III (7.3%), biotype IV (7.3%) and biotype V (7.3%). Our study revealed that the deft index was significantly higher in children who were biotype I carriers than in those who were nonbiotype I carriers. This observation suggests that biotype I may be related to the higher incidence of dental caries in our preschool children.

Molecular and epidemiologic study of multiresistant Escherichia coli infections in Kaohsiung area.

Totally 70 Escherichia coli (E. coli) isolated strains from patients in Chang Gung Memorial Hospital, Kaohsiung Medical College affiliated Chung Ho Memorial Hospital and the Navy General Hospital of the Republic of China, all in Kaohsiung, were collected during the period of July 1986-August 1987 and examined for their susceptibility to several antimicrobial agents as well as for their plasmid contents. The isolates tested showed a high level of resistance to ampicillin (Ap) (90.4%), piperacillin (Pip) (89.0%), tetracycline (Tc) (88.7%), streptomycin (Sm) (87.7%), chloramphenicol (Cm) (78.4%), kanamycin (Km) (72.6%) and sulfamethoxazole/trimethoprim (Sxt) (54.2%). Observations of the resistance patterns revealed that the dominant type was Ap-Tc-Cm-Sm-Pip-Km. The results of plasmid curing and transfer experiments indicated that three plasmids with molecular weights 49.2-51.6 Mdal, 69.8-178 Mdal and a third one with molecular weight larger than 178 Mdal carried resistance determinants for Gm-Nn, Sm-Km-Tc and Tc-Cm-Km, respectively.

Cytotoxicity of isoquinoline alkaloids and their N-oxides.

A series of 53 isoquinoline alkaloids and their N-oxides have been tested for their cytotoxicity against A-549, HCT-8, KB, P-388, and L-1210 cells. These alkaloids include two tetrahydroprotoberberines, two protoberberines, six aporphines, one morphinandienone, five oxoaporphines, seven phenanthrenes, one spirobenzylisoquinoline N-oxide, nine aporphine N-oxides, seven benzyltetrahydroisoquinoline N-oxides, one benzylisoquinoline N-oxide, one protopine N-oxide, three tetrahydroprotoberberine N-oxides, four pavine N-oxides, and four phenanthrene N-oxides. The results are discussed on the basis of structure-activity relationships.

Screening of isoquinoline alkaloids and their derivatives for antibacterial and antifungal activities.

A screening test of antimicrobial activities for some of the isoquinoline alkaloids and their first and second Hofmann elimination products was conducted in this study. Results showed that (+)-actinodaphnine (1), (+)-N-Me-actinodaphnine (2), (+)-anonaine (17), (-)-xylopine (19) and (-)-N-Me-xylopine MeI (20), had the strongest inhibitory activities against three G(+) bacteria (Bacillus cereus, Micrococcus sp. and Staphylococcus aureus) MIC greater than or equal to 50 micrograms/ml). Whereas anhydroushinsunine (34), anhydroushinsunine MeI (35), ushinsunine isomethine (38), dicentrine methine (23), roemerine methine (26), dicentrine bismethine (29) and O-Me-armepavine methine (48) also had antibacterial effects against these same three G(+) bacteria (MIC = 50-300 micrograms/ml). However, only (+)-actinodaphnine (1) and roemerine methine (26) showed weak effects against two G(-) bacteria (Escherichia coli and Klebsiella pneumonia) (MIC = 300 micrograms/ml). (+)-Actinodaphnine(1), (+)-N-Me-actinodaphnine (2), (+)-anonaine (17), anhydroushinsunine (34), anhydroushinsunine MeI (35), ushinsunine isomethine (38), O-Me-armepavine methine (48) and O,O-di-Et-N-Me-coclaurine methine (49) had potent antifungal activities against Candida albicans, Cryptococcus neoformans and other Candida species (MIC = 62.5-1000 micrograms/ml).

[Evaluation of a self-prepared enzyme immunoassay kit for detection of alpha-fetoprotein].

A "sandwich" technique of enzyme immunoassay (EIA) kit for human alpha-fetoprotein has been developed by using commercially available reagents. The assay range is 0.5-60 ng/ml. Within-run coefficients of variation (C.V.) for 20 determination at three different concentrations were 4.4-10.3%, and between-run C.V. were 4.9-10.8%. The sensitivity and specificity of the kit has been compared with those of two commercially available kits (Abbott and Roche r1 = 0.991, r2 = 0.989.) This kit has some advantages over currently available kits. The assay is incubated in room temperature, no waterbath is required. The solid phase is dry store, it have long shelf life. The kit is inexpensive to operate, and it is suitable for any laboratory.

The effects of genkwadaphnin and gnidilatidin on the growth of P-388, L-1210 leukemia and KB carcinoma cells in vitro.

Daphnane diterpene esters have previously been shown to have antineoplastic activity in vivo against the growth of P-388 lymphocytic leukemia cells. These studies demonstrate cytotoxic activity of genkwadaphnin and gnidilatidin against P-388 lymphocytic leukemia, L-1210 lymphoid leukemia and human KB carcinoma cell growth in vitro. At the ED50 values in the respective tumor lines DNA synthesis was preferentially suppressed in all three cell lines. RNA synthesis was essentially unaffected by the agents. Protein synthesis inhibition by the two agents demonstrated selectivity, e.g. in P-388 cells significant inhibition, in L-1210 cells marginal inhibition and in KB cells no inhibition was observed at these concentrations. Multiple sites in DNA synthesis were found to be inhibited by the daphnane diterpene esters. Two to three times the ED50 concentration in the respective tumor lines was required to observe suppression of DNA synthesis. Purine de novo synthesis appeared to be the major site of inhibition, with inosine monophosphate dehydrogenase and phosphoribosyl pyrophosphate amido transferase activities being inhibited in all three tissue lines. Dihydrofolate reductase activity was inhibited, significant only in the P-388 and KB cells. The magnitude of the enzyme suppression by the agents varied with the tumor line. However, the degree of enzyme suppression was of sufficient magnitude to account for the observed purine and DNA synthesis inhibition by the daphnane diterpene esters.

Antitumor agents LIX: effects of quassinoids on protein synthesis of a number of murine tumors and normal cells.

The quassinoids (brusatol, bruceantin, bisbrusatolyl esters, and bisbruceantinyl esters of succinic and malonic acids) were observed not to be universal protein synthesis inhibitors. Rather, they were selective for both the types of cancers, e.g., P-388 lymphocytic leukemia, Ehrlich and hepatoma carcinoma and L-1210 lymphoid leukemia, as well as types of normal tissues (e.g., lymphocytes), in which they demonstrated protein synthesis inhibition. The data suggest that the observed difference in the magnitude of protein synthesis inhibition of two P-388 lymphocytic leukemia cell lines by the quassinoids was at the ribosomal levels, whereas the observed difference in normal livers from various strains of mice involve differences in cell membrane transport of the quassinoids into the various tissues. Detailed studies indicated that the mode of action of the quassinoids as protein synthesis inhibitors was identical in all of the cells where inhibition was observed; i.e., the elongation step of protein synthesis was blocked by the quassinoids. The data derived from assays for polyuridine-directed polyphenylalanine synthesis of isolated ribosomes demonstrated that the ID50 values obtained were consistent with the observed inhibition of whole cell protein synthesis inhibition for P-388 cells, as well as for BDF1 and DBA/2 liver cells. The ID50 values obtained from various cells, e.g., P-388 cells and normal liver, were all in the microM range and are consistent with previously published values for the quassinoids in the rabbit reticulocyte and yeast systems.

Investigation of sesquiterpene lactones as protein synthesis inhibitors of P-388 lymphocytic leukemia cells.

The mode of action of helenalin and bis(helenalinyl) malonate as protein synthesis inhibitors of P-388 lymphocytic leukemia cells was investigated. The initial characterizations were carried out in crude lysates of the P-388 cells. In the lysate, there was a 4 min lag after the addition of drug before inhibition of protein synthesis occurred. Both drugs allowed run-off of preformed polysomes, but did significantly inhibit the formation of the 80 S initiation complex suggesting a preferential inhibition of one or more initiation reactions. The effect of these drugs on inhibition of both elongation and initiation reactions was further investigated using more fractionated systems prepared from P-388 cells. Poly(U)-directed polyphenylalanine synthesis was marginally inhibited by both drugs, but the degree of inhibition was not sufficient to explain the inhibition observed in either the lysate or in whole cell preparations of P-388. The formation of the ternary initiation complex was not significantly inhibited by either drug, but the conversion of this complex to the 48 and 80 S initiation complexes was inhibited. The inhibition of 48 S initiation complex formation by both drugs was sufficient to explain their inhibition of protein synthesis in whole cells.

Antitumor agents LVI: the protein synthesis inhibition by genkwadaphnin and yuanhuacine of P-388 lymphocytic leukemia cells.

Two natural products isolated from the plant Daphne genkwa have been shown to possess antileukemic activity in mice. Genkwadaphnin and yuanhuacine were observed to inhibit DNA and protein synthesis in P-388 leukemic cells. A detailed study of the effects of these two diterpene esters on protein synthesis of leukemic cells was undertaken. The major effects of genkwadaphnin and yuanhuacine on protein synthesis were blockage of the elongation process and interference with the peptidyl transferase reaction. The latter reaction was suppressed at concentrations of the diterpene esters which were commensurate with concentrations that inhibited whole cell in vitro protein synthesis in P-388 cells.

Antitumor agents LII: The effects of molephantinin on nucleic acid and protein synthesis of Ehrlich ascites cells.

Molephantinin, a germacranolide, has previously been shown to possess antineoplastic activity in rodents. The principle effect of molephantinin on Ehrlich ascites carcinoma cells was to depress DNA and protein synthesis both in vivo and in vitro. DNA synthesis was inhibited at the following sites: DNA polymerase, purine synthesis specifically at inosinic acid dehydrogenase and to a lesser degree at dihydrofolate reductase, pyrimidine synthesis at orotidine monophosphate decarboxylase, thymidine kinase, histone phosphorylation, and oxidative phosphorylation processes. The protein synthesis inhibition pattern resembled more an initiation inhibitor as opposed to an elongation inhibitor.

Antitumor agents XLVIII: Structure-activity relationships of quassinoids as in vitro protein synthesis inhibitors of P-388 lymphocytic leukemia tumor cell metabolism.

A series of brusatol, bisbrusatol, and bruceantin esters were examined for their ability to inhibit protein synthesis in P-388 lymphocytic leukemia cells. Compounds which produced high T/C % values (170-272) resulted in ID50 of 5.4-15.5 microM for inhibition of whole cell protein synthesis, ID50 of 1.3-13 microM for inhibition of endogenous protein synthesis in cell homogenates, and ID50 of 1.9-6 microM for inhibition of polyuridine directed polyphenylalanine synthesis using "runoff" ribosomes and a "pH 5" enzyme preparation. The polyuridine directed polyphenylalanine synthesis requires neither initiation nor termination factors, suggesting that quassinoids are exclusively elongation inhibitors. Bruceantin, brusatol, and bisbrusatolyl malonate allowed a runoff of the polyribosomes to 80S free ribosomes. However, formation of the ternary complex and 80S initiation complex were not inhibited by the quassinoids. Thus, these agents do not affect the individual steps leading to the formation of a stable 80S initiation complex in P-388 cells. Brusatol, bruceantin, and bisbrusatolyl malonate inhibited the formation of the first peptide bond between puromycin and [3H]methionyl-transfer RNA bound to the initiation complex, indicating peptidyl transferase activity is inhibited by the quassinoids in P-388 cells. These studies also suggest that the free 80S ribosome is the site of binding by the quassinoid. Ribosomes actively conducting protein synthesis will continue protein synthesis and terminate before the quassinoids bind. This proves quassinoids are elongation inhibitors of tumor cells. A strong correlation was observed between potent antileukemic activity and the ability to inhibit protein synthesis in P-388 lymphocytic leukemia cells.

Antitumor agents XLVI: In vitro effects of esters of brusatol, bisbrusatol, and related compounds on nucleic acid and protein synthesis of P-388 lymphocytic leukemia cells.

A series of esters of brusatol, bisbrusatol, and bruceantin were shown to have potent antileukemic activity. Antineoplastic activity was correlated with the ability of the compounds to suppress DNA and protein synthesis in P-388 lymphocytic leukemia cells. Compounds with high T/C% values successfully inhibited DNA polymerase activity and purine synthesis. The ability to inhibit protein synthesis during the elongation process also correlated positively with high antileukemic activity in this series of quassinoids. Dihydrofolate reductase activity and basal and adenosine diphosphate stimulated respiration of P-388 cells were also inhibited.

Antitumor agents XLVII: The effects of bisbrusatolyl malonate on P-388 lymphocytic leukemia cell metabolism.

Bisbrusatolyl malonate, which was shown previously to be active against P-388 lymphocytic leukemia cell growth, was investigated for inhibitory effects on nucleic acid and protein synthesis. DNA and RNA synthesis as well as protein synthesis were markedly inhibited at 10,25, and 50 mu mole final concentrations in vitro. The major sites of inhibition of nucleic acid synthesis appeared to be DNA polymerase, messenger and transfer RNA polymerases, orotidine-5'-monophosphate decarboxylase, phosphoribosyl pyrophosphate amino transferase, and dihydrofolate reductase. Moderate inhibition of nucleotide kinase activities and oxidative phosphorylation processes occurred after drug treatment. Cyclic adenosine monophosphate levels were reduced. Protein synthesis was inhibited during the elongation step of peptide synthesis. The data suggested that bisbrusatolyl malonate interfered with the peptide bond formation. However, the ongoing polypeptide synthesis must be completed before the drug can bind to the ribosome effectively.

Antitumor agents XLII: Comparison of antileukemic activity of helenalin, brusatol, and bruceantin and their esters on different strains of P-388 lymphocytic leukemic cells.

Based on the fact that some known antineoplastic agents possess an ester moiety within their structure, the esters of helenalin, a sesquiterpene lactone, and of brusatol and bruceantin, quassinoids, were synthesized and tested for antileukemic activity in the P-388 screen. These agents gave different T/C% values dependent on the P-388 lymphocytic leukemia strain and the host strain of mice used. Later studies demonstrated that the agents caused different degrees of inhibition of nucleic acid and protein synthesis in the various P-388 strains. The higher the degree of inhibition of precursor incorporation into the nucleic acid or protein, the higher was the T/C% value obtained in a given P-388 strain. The study demonstrates the lack of consistency of P-388 lymphocytic leukemia cell lines used in various laboratories and indicates that the inbred strain of mice is a critical factor in the tolerance of drug toxicity and, thus, T/C% obtained.

Inhibition by thiopeptin of bacterial protein synthesis.

Thiopeptin, a sulfur-containing antibiotic, was found to inhibit protein synthesis in a bacterial ribosomal system. The pretreatment of ribosomal subunits with the antibiotic revealed that thiopeptin may act on the 50 S ribosomal subunit. The elongation of peptide chain on the ribosome is more profoundly blocked by the antibiotic than the initiation of protein synthesis. It was demonstrated that thiopeptin inhibits elongation factor (EF)-Tu-dependent GTP hydrolysis and binding of aminoacyl-tRNA to the ribosome. The peptidyl transferase-catalyzed puromycin reaction is not significantly affected by the antibiotic. Thiopeptin inhibits EF-G-associated GTPase reaction, and translocation of peptidyl-tRNA and mRNA from the acceptor site to the donor site. Protein synthesis in ribosomal systems, obtained from rat liver and rabbit reticulocytes are insensitive to the antibiotic.