Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-γ-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects off MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N⁵-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N⁵-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.     

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12

Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.     

Infrared spectra of carbon monoxide complexes of indoleamine 2,3-dioxygenase and L-tryptophan 2,3-dioxygenases. Effects of substrates on the CO-stretching frequencies

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited two CO stretch bands at 1953 and 1933 cm-1 with half-band widths (delta v 1/2) of both approximately 15 cm-1. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm-1 with the delta v 1/2 of 15 cm-1. Carbonmonoxy L-tryptophan 2,3-dioxygenase of Pseudomonas acidovorans in the absence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965 and 1958 cm-1 (delta v 1/2 for the fused band; 25 cm-1), which was converted into a sharp single band at 1968 cm-1 (delta v 1/2; 10 cm-1) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver L-tryptophan 2,3-dioxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm-1. By the addition of L-tryptophan, the spectrum changed into that with two distinct bands at 1972 and 1920 cm-1 (delta v 1/2; 6 and 13 cm-1, respectively). These spectra were insensitive to pH in a range where the enzymes were not denatured (neutral to near pH 9). The infrared spectra of the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and alpha-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturating amount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.     

Congenital absence of flexor pollicis longus without hypoplasia of thenar muscles

A 7-year-old girl who was unable to flex her left thumb at the interphalangeal joint proved to have an extremely hypoplastic flexor pollicis longus with normal thenar muscles, which is very rare. The flexor digitorum superficialis of the ring finger was transferred to the inserting portion of the flexor pollicis longus tendon with good results. The patient's cooperation seems to be a factor determining the prognosis.     

Bile acid metabolism in partially hepatectomized rats

The bile flow and the bile acid secretion, calculated on liver weight basis, increased 12 H and 24 H after 60-70% hepatectomy and returned to the initial levels thereafter. The biliary phospholipid secretion much more increased than bile acids, but the cholesterol secretion decreased. Bile acid composition changed with an increase of the cholic acid group and a decrease of the chenodeoxycholic acid group in both bile and feces. These changes almost disappeared on Day 14. The pool size of bile acid decreased maximally on Day 4 to about 40% of the initial, but the distribution of bile acids in the enterohepatic circulation was not changed. The fecal cholesterol and coprostanol markedly decreased on Day 2 but gradually returned to the initial levels according to the recovery of diet intake. The fecal bile acids decreased on Day 2, increased on Day 4, and returned to the normal range after Day 7. In conclusion, the regenerating liver secretes more bile, bile acids and phospholipids, and less cholesterol than the normal liver. Cholic acid predominates in the bile acids. These changes restored to the initial levels by about one week after the operation.     

Mammalian signal peptidase: partial purification and general characterization of the signal peptidase from microsomal membranes of porcine pancreas

Signal peptidase has been enriched extensively from microsomal membranes of porcine pancreas. Microsomal membranes were washed with 1 M KCl and Brij 35, and then solubilized with 1% Nonidet P-40. The solubilized signal peptidase was purified by DEAE-cellulose chromatography and Sepharose CL-6B filtration. Cleavage of pre-human placental lactogen with the partially purified enzyme gave the mature form, whose NH2-terminus was identified as valine. The signal peptidase is heat-labile and approximately 90% of the enzymatic activity was lost at 60 degrees C within 1 min. The pH optimum of the activity was 7 to 8. Chymostatin and o-phenanthroline at concentrations of 2.5 mM inhibited the signal peptidase activity by 62% and 30%, respectively.     

Antigen-specific murine T-cell lymphomas. II. H-2 restriction of primary and secondary responses

A stable clone of C57BL/6 (H-2b) radiation leukemia virus transformed ovalbumin (OVA)-specific murine T-cell lymphoma cells was able to mediate carrier-specific helper activity. The ability of these lymphoma cells to express helper activity for both primary and secondary hapten-specific B-cell responses was analyzed in nonirradiated normal or hapten-primed recipients. The lymphoma cells augmented anti-hapten responses in a carrier-specific manner; no bystander effects were noted. Helper activity was primarily noted in the IgG responses. The genetic restrictions affecting the expression of lymphoma-mediated helper activity were also analyzed. The pattern of restriction indicated that genes in the H-2 complex controlled the expression of helper activity; disparities at the Igh complex failed to influence helper activity. The cellular site of the H-2 restriction was between the antigen-presenting cells and the T-cell lymphoma not between the T and B cells. Precise intra-H-2 mapping of the gene(s) which control expression of lymphoma-mediated helper activity was attempted. Although most of the data were consistent with localization of the gene(s) to the I-A region, anomolous responses were noted in one strain.     

Molecular and enzymatic properties of 1,2-α-d-mannosidase fromAspergillus saitoi

The molecular properties, such as molecular weight, N-and C-terminal amino acids, amino acid composition, and circular dichroism, of 1,2--mannosidase isolated from the culture filtrate ofAspergillus saitoi were determined.The enzyme had aK _m of 0.67 mM andk _cat of 1.27/s with mannobiose at pH 50.0 and 30°C. The anomeric configuration of the reaction products of the enzyme was examined by studying the -anomer. A single Manl2Man linkage in intact Taka-amylase A fromAspergillus oryzae was hydrolyzed, producing free mannose.     

Effect of Bordetella pertussis leukocytosis (lymphocytosis)-promoting factor (LPF) on the physical lymphoepithelial-cell association studied with the use of an in vitro model of mouse thymus

The effect of highly purified leukocytosis (lymphocytosis)-promoting factor (LPF) of Bordetella pertussis on physical lymphocyte and reticuloepithelial (RE) cell association was studied in an in vitro thymus model. First, a simplified in vitro system to assess the lympho-RE-cell association was developed. A completely confluent layer of thymic RE cells was formed by cultivating trypsinized thymus cell suspensions from 2- to 7-day-old mice. When thymic lymphoid cells were seeded on this cell layer and cultivated overnight, a significant proportion of them were found underneath the RE cell layer. This physical lympho-RE-cell association was quantitated by counting the lymphoid cells underneath the RE cell layers. Second, the effect of LPF on this physical lympho-RE-cell association phenomenon was investigated. Addition of LPF to the culture markedly inhibited the formation of the lympho-RE-cell complex; that is, it inhibited the infiltration of lymphoid cells under the RE cell layer. LPF rendered a nearly maximal level of inhibitory effect at a dose of 0.1 ng/ml. Furthermore, LPF enhanced the liberation of lymphoid cells from preformed lympho-RE-cell complexes. On the other hand, LPF had no direct cytotoxic effect on lymphoid cells at doses below 1 microgram/ml. In order to investigate whether LPF produced the effect by acting on lymphoid cells, RE cells, or both, the following experiments were performed. When lymphoid cells were pretreated with LPF and added to normal RE cell layers, the lympho-RE-cell association was maximally inhibited above the dose of 1 ng/ml. Treatment of these LPF-treated lymphoid cells with anti-LPF antibodies failed to abrogate the effect of LPF. When RE cell layers were similarly pretreated with LPF and were cultivated with normal lymphoid cells, however, much higher doses of LPF, above 100 ng/ml, were required for maximal inhibition. Furthermore, treatment of these LPF-treated RE cells with anti-LPF antibodies abrogated the effect of LPF. Therefore, the apparent effect of LPF on RE cells was considered to be due to the carry-over by RE cells of LPF, which should directly act on lymphoid cells at extremely low doses. On the basis of these results, it was concluded that LPF acted directly on lymphoid cells without mediation of RE cells. These in vitro results appear to parallel the effects of LPF in vivo, where it induces a depletion of cells in the thymus. The model may be useful to study this phenomenon and the concomitant accumulation of blood lymphocytes.     

Twinning rate in spontaneous abortions.

An unselected series of spontaneous abortions and their mothers were karyotyped with Q-bands to obtain a frequency of twin conceptions lost during the first trimester. Among 661 spontaneous abortions, 15 twin pairs were identified including two sets of conjoined twins. Analysis of Q-band variants permitted the exclusion of cases with two cell lines that could be attributed to maternal contamination or mosaicism. The twinning rate among spontaneous abortions was 1/44 compared with 1/103 live births and stillbirths in the Ontario population. If Weinberg's differential method is applied to these data, the frequency would be as high as 1/30 under the assumption that the incidence of monozygotic twins among abortions is the same as that for live births.