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.     

An immobilized fork as a termination of replication intermediate in Bacillus subtilis

The structure of a DNA intermediate associated with termination of chromosome replication in Bacillus subtilis and derived from a unique BamHI 24.8 X 10(3) base-pair (bp) region of the chromosome has been investigated. The intermediate has properties expected for a forked structure. Gel electrophoresis followed by Southern transfer and hybridization to cloned DNA has shown it to comprise single strands of 15.4 X 10(3) bp and 24.8 X 10(3) bp, in approximately equimolar amounts. After purification away from the bulk of chromosomal DNA, electron microscopy of the intermediate established that 15% of the DNA was present as branched molecules and a significant proportion (11 of 31) of these contained two arms of matching length. The average dimensions (best estimates) of this unique class of Y-shaped molecule were 9.5(+/- 0.3) X 10(3), 15.1(+/- 0.4) X 10(3) and 24.6 24.6(+/- 0.6) X 10(3) bp for the stem, arms and end-to-end length, respectively. These values are consistent with the single strand composition of the intermediate as found. Furthermore, hybridization of the single strands to DNA from known locations within the BamHI 24.8 X 10(3) bp region has established the orientation of the forked intermediate relative to the genetic map. The intermediate presumably reflects the immobilization of the clockwise replication fork within the 24.8 X 10(3) bp region, at a location approximately 15.4 X 10(3) bp from the right end.     

Synaptosomal Calcium Metabolism During Hypoxia and 3,4-Diaminopyridine Treatment

A decline in the calcium-dependent release of neurotransmitters appears to underlie the decreased neuronal function that accompanies reduced oxygen tensions (hypoxia). To determine if alterations in calcium uptake are primary to these changes, synaptosomal calcium uptake was measured in the presence of 100%, 2.5%, or 0% oxygen. Calcium uptake declined 60.2 +/- 0.1 and 82.4 +/- 2.5% with 2.5% and 0% when compared with 100% oxygen, respectively. 3,4-Diaminopyridine stimulated calcium uptake by synaptosomes when they were incubated in low-potassium media. It also diminished the hypoxic-induced decline in calcium uptake to 30.6 +/- 3.1 and 33.5 +/- 3.1% with 2.5% and 0% oxygen, respectively. External binding to the synaptosomal plasma membrane declined to 29.2 +/- 0.3 or 11.8 +/- 0.9% when the oxygen tension was reduced to 2.5% or 0% oxygen. 3,4-Diaminopyridine increased this superficial binding from 111.7 +/- 0.3 to 86.5 +/- 0.9 or 23.4 +/- 0.9% with 100%, 2.5%, or 0% oxygen when compared with 100% oxygen without 3,4-diaminopyridine, respectively. Thus, the decline in neuronal processing that accompanies acute hypoxia may be due to altered calcium homeostasis, which diminishes neurotransmitter release.     

The incorporation of J chain into reassembled human immunoglobulin M

Human IgM (immunoglobulin M) was reduced with 24mm-mercaptoethylamine. This atreatment resulted in complete dissociation to IgMs subunits and free J chain. Intr-subunit interchain disulphide bonds remained intact. The mixture then was encouraged to reoxidize. The schlieren pattern of the reoxidized mixture showed the presence of a considerable quantity of IgM in addition to residual IgMs. The isolated reassembled IgM did not dissociate in 5m-guanidinium hydrochloride. It apparently contained the same amount of covalently attached J chain as did native IgM. The J chain was a part of the high-molecular-weight Fc fragment obtained from the reassembled IgM.     

Ribonucleic Acid Synthesis During the Differentiation of Sporangia in the Water Mold Achlya

The role of ribonucleic acid (RNA) synthesis in the development of sporangia in the saprolegniaceous mold Achlya was studied. Methods were developed for growing and treating large populations of mycelia so that the hyphal tips would differentiate into sporangia with considerable synchrony. Under the starvation conditions imposed for the differentiation of sporangia, net RNA, deoxyribonucleic acid (DNA), and protein synthesis ceased. However, incorporation of radioactive precursors into RNA continued at a high rate throughout the period of differentiation, showing that the enzymatic mechanism for RNA synthesis was still in an active state. Actinomycin D inhibited the differentiation of sporangia and the incorporation of labeled precursors into RNA. The level of actinomycin used did not inhibit the normal outgrowth and branching of the mycelia that occurred during differentiation. Thus, DNA-dependent RNA synthesis was required for the differentiation of sporangia. Sucrose gradient analysis of newly synthesized RNA showed that only the ribosomal and soluble fractions of RNA were labeled during vegetative growth. During the differentiation of sporangia, ribosomal and soluble RNA fractions were also labeled, and, in addition, a heterodisperse fraction of labeled RNA which was heavier than ribosomal RNA appeared; this fraction was not evident in the newly synthesized RNA from vegetative mycelia.