Effects of diethyldithiocarbamate (ddtc) on the plasma biotransformations of tetrachloro(d,i-trans)-1,2-diaminocyclohexaneplatinum(iv) (tetraplatin) in fischer 344 rats

We have studied the effects of diethyldithiocarbamate (DDTC) on the biotransformations of toxic doses of tetrachloro (d,l-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in Fischer 344 rats. In animals not treated with DDTC, tetraplatin was rapidly converted to dichloro(d,I-trans)1,2-diaminocyclohexaneplatinum(II) [PtCl₂(dach]. Subsequent biotransformations included the transient formation of the (d,I-trans)1,2-diaminocyclohexane-aquachloroplatinum(II) [Pt(H₂O)(Cl)(dach)]+ complex, followed by formation of the platinum (Pt)-methionine and either Pt-cysteine or Pt-ornithine complexes. Significant amounts of free (d,I-trans) 1,2-diaminocyclohexane (dach) were observed in plasma as a result of intracellular trans-labilization reactions. DDTC caused a marked decrease in both total and protein-bound platinum in the circulation. A significant increase in the plasma concentration of free dach was also observed as a result of formation of the Pt(DDTC)₂ complex. Some of the free dach could have arisen from intracellular reactions with DDTC, but the displacement of platinum from plasma proteins was more than sufficient to account for the increase in free dach in the circulation. DDTC treatment also decreased plasma concentrations of tetraplatin, PtCl₂(dach), [Pt(H₂O)(Cl)(dach)]⁺, the Pt-methionine complex, and one unidentified biotransformation product, but had no effect on the Pt-cysteine (or Pt-ornithine) complex. These effects of DDTC on protein-bound platinum and low-molecular-weight biotransformation products in plasma may contribute to the decrease in tetraplatin toxicity seen in DDTC-treated rats.     

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.     

Direct sequencing and bidirectional allele specific polymerase chain reaction of the bovine beta-casein B variant.

We have used the polymerase chain reaction (PCR) to amplify exon VII of the bovine beta-casein gene. The mutations responsible for the B variant were identified by direct sequencing of the amplification products. A bidirectional allele-specific PCR method (BAS-PCR) has been developed using oligonucleotides overlapping the mutation site at their 3' ends. This new procedure allows a rapid and reliable discrimination between the B and non-B alleles of beta-casein.     

Methods in clinical hemorheology: the continuous measurement of arterial blood density and blood sound speed in man

Both blood density and sound speed are closely related to total protein concentration in blood and, as a consequence, to rheologically important parameters of blood. Two methods that permit continuous measurement of these properties, the mechanical oscillator technique and the new ultrasonic technique, were used for measuring blood protein concentration over a continuous period of time in a group of hemodialysis patients and in volunteers. It was seen that the concentration of the components of blood varies considerably. This variability is related to transport phenomena within as well as to the flow of masses across the cardiovascular compartment. From the continuous measurement of concentrations during hemodialysis treatment, relative changes in blood volume can be recorded in order to control the fluid balance of the patient. Rapid fluctuations at the macroscopic scale with periods of 5 to 30 seconds are due to heterogeneities at the microscopic scale and to the particular rheological behaviour of the red blood cells at the level of the capillaries and the small blood vessels. The amplitude of rapid oscillations increased up to 1.2% in terms of hematocrit values when there was rhythmic, spontaneous breathing at various frequencies. The measurement of concentrations at an accessible measuring site may be used to investigate the rheology of blood in the human microvasculature.     

High sequence similarity within ras exons 1 and 2 in different mammalian species and phylogenetic divergence of the ras gene family

We have determined the canine and feline N-, K-, and H-ras gene sequences from position +23 to +270 covering exons I and II which contain the mutational hot spot codons 12, 13, and 61. The results were used to assess the degree of similarity between ras gene DNA regions containing the critical domains affected in neoplastic disorders in different mammalian species. The comparative analyses performed included human, canine, feline, murine, rattine, and, whenever possible, bovine, leporine (rabbit), porcelline (guinea pig), and mesocricetine (hamster) ras gene sequences within the region of interest. Comparison of feline and canine nucleotide sequences with the corresponding regions in human DNA revealed a sequence similarity greater than 85% to the human sequence. Contemporaneous analysis of previously published ras DNA sequences from other mammalian species showed a similar degree of homology to human DNA. Most nucleotide differences observed represented synonymous changes without effect on the amino acid sequence of the respective proteins. For assessment of the phylogenetic evolution of ras gene family, a maximum parsimony dendrogram based on multiple sequence alignment of the common region of exons I and II in the N-, K-, and H-ras genes was constructed. Interestingly, a higher substitution rate among the H-ras genes became apparent, indicating accelerated sequence evolution within this particular clade. The most parsimonious tree clearly shows that the duplications giving rise to the three ras genes must have occurred before the mammalian radiation. Received: 23 July 1997 / Accepted: 30 October 1997