The effect of CaCl2 and verapamil on the binding of cisplatin to cultures of normal and transformed human fibroblasts

Normal and transformed human fibroblasts were treated for either 1 sec or 1 h with the antitumor drug cis-dichlorodiamine platinum (cisplatin). The dose response of drug binding and cell survival was determined for cells treated with the drug in the presence or absence of 3.0 mM CaCl2. The levels of drug initially bound to both cell types was similar and was not affected by the presence of Ca2+. The dividing non-transformed cells were most sensitive to killing by short treatment with cisplatin compared to the transformed cells or the confluent non-transformed cultures. After 1 h of cisplatin treatment, the levels of drug bound to the cells were significantly less than that recovered after the shorter treatment. This time-dependent loss of cisplatin was inhibited both by CaCl2 and by the calcium channel blocking agent, verapamil. The higher levels of cisplatin bound after 1 h in the presence of these agents, however, did not in all cases result in decreased survival; the effects were dependent on cell type and on whether the cells were dividing or confluent. Analysis of cisplatin binding to cell cultures indicated that initially the cisplatin was weakly attached to the pericellular and substratum attached material but that with time, the drug bound to this material decreased. This time-dependent removal from the extracellular matrix was much less in the transformed cell cultures and was inhibited by calcium. We propose that the major site of interaction of cisplatin with these cells is in the extracellular matrix and with time the cultures alter their extracellular matrix to decrease this binding. This removal process appears to involve calcium or calcium transport since CaCl2 and verapamil both block these changes.     

Oxysterols: biological activities and physicochemical studies.

To improve the understanding of the various biological activities of oxysterols (oxygenated derivatives of cholesterol), studies of their physicochemical properties have been undertaken. Oxysterols modify membrane dynamic properties which consequently trigger several biological effects. Despite the presence of at least one oxygenated group in addition to the C3 beta-hydroxyl, oxysterols insert perfectly into the lipidic bilayer of the membrane inducing a condensing effect similar to, but less potent than, that of cholesterol. In biological membranes oxysterols probably interact with membrane components as they are not easily exchanged after their incorporation into the cell membrane. These lipid-protein interactions are probably crucial for the expression of the biological activities of the oxysterols.     

Quantification of short term signaling by the epidermal growth factor receptor.

During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15-30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.     

Behavioural discrimination among chromosomal races of the house mouse (Mus musculus domesticus)

This work is concerned with the extent of behavioural discrimination between three chromosomal races of the house mouse (the standard 40-chromosome race and a 32- and 36-chromosome races) found in the vicinity of a hybrid zone in northern Scotland. Mice were investigated for several elements of their social behaviour. Within-population dyadic encounters did not show consistent behavioural differences attributable to karyotype among five populations (two standard race, two 36-chromosome race, one 32-chromosome race). Between-population dyadic encounters revealed significant differences between three populations. The standard population examined appeared to be the most “open” to foreigners, the 32-chromosome population the most “closed” while the 36-chromosome mice displayed an intermediate response. Differences in behaviour displayed during between-population as compared to within-population dyadic encounters revealed the occurrence of behavioural discrimination between populations. The implication of these results on the dynamics of the hybrid zone are discussed.     

The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.     

A simple method to test condoms for penetration by viruses.

A method by which virus penetration through condoms can be tested with simple, inexpensive equipment is described. The method uses chi X174 bacteriophage as the challenge virus and physiologically relevant pressure. Penetration by 0.1 microliters (or less) of challenge suspension can be readily detected. As examples, latex and natural-membrane condoms were examined.