Effect of volume expansion on hemodynamic variables in nephrectomized dogs

We have previously demonstrated that blood pressure elevation by acute blood volume expansion is volume-dependent during the infusion period and resistance-dependent in the post-infusion period in normal anesthetized dogs, and that such an increase in blood pressure is associated with a potentiation of the pressor response to norepinephrine. To evaluate the possible renal contribution to these hemodynamic changes, blood volume expansion was performed for 1 h with dextran dissolved in lactated Ringer's solution (20 ml/kg) in 15 nephrectomized dogs. The mean blood pressure, cardiac output and total peripheral resistance at the end of infusion were 126%, 225% and 60%, respectively; 3 h after volume expansion they were 126%, 151%, and 92% respectively. However, in 4 dogs, there was an increase in mean blood pressure (138%) 3 h after volume expansion. This was thought to result from an increase in the total peripheral resistance (133%) associated with the recovery of cardiac output (106%). The pressor response to norepinephrine (0.5 microgram/kg) was potentiated after volume expansion. These results indicate that the handling of volume by the kidney contributed to the maintenance of an elevated level of cardiac output. However, nephrectomy did not seem to interfere with the hemodynamic switching of the causative factor for blood pressure elevation from increased cardiac output to increased total peripheral resistance. Neither was the potentiation of pressor response to norepinephrine affected.     

Reassessment of plasma angiotensins measurement: effects of protease inhibitors and sample handling procedures.

Characterization of C- and N-terminal forms of angiotensin (Ang) peptides mandated assessment of methods to determine plasma levels. 125I-Ang I, 125I-Ang II, and 125I-Ang(1-7) were added to blood samples in the presence of protease inhibitors. Ethylenediaminetetraacetic acid (EDTA) inhibited the conversion of 125I-Ang I to 125I-Ang II. o-Phenanthroline and EDTA (EDTA + o-Ph) did not eliminate [des-Asp1] fragments or 125I-Ang(1-7). The combination of EDTA + o-Ph and pepstatin A or 4-(chloromercuri) benzoic acid (PCMB) significantly reduced 125I-Ang(1-7) generation. Only PCMB plus EDTA + o-Ph eliminated [des-Asp1] fragments. Authentic plasma values of Ang peptides require the correct choice of protease inhibitors.     

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.     

Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome.

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.     

Two genes controlling acute phase responses by the antitumor polysaccharide,lentinan

Lentinan, a -1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) andMus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence lenght polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 andltn1.2) responsible for DT-APR.ltn1.1 is closely linked toD3Mit11 on chromosome 3 andltn1.2 toD11Nds9 on chromosome 11 (P<0.001). The linkage analysis also suggested thatltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that theltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.     

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.     

Alteration of N-terminal residues of mature human lysozyme affects its secretion in yeast and translocation into canine microsomal vesicles

Signal sequences play a central role in the initial membrane translocation of secretory proteins. Their functions depend on factors such as hydrophobicity and conformation of the signal sequences themselves. However, some characteristics of mature proteins, especially those of the N-terminal region, might also affect the function of the signal sequences. To examine this possibility, several mutants of human lysozyme modified in the N-terminal region of the mature protein were constructed, and their secretion in yeast as well as in vitro translocation into canine pancreatic microsomes were analyzed using an idealized signal sequence L8 (MR(L)8PLAALG). Our results show the following. (1) Change in the charge at the N-terminal residue of the mature protein does not affect secretion drastically. (2) Substitution of a proline residue at the N terminus prevents cleavage of the signal sequence, although translocation itself is not impaired. (3) Excessive positive charges in the N-terminal region delay translocation of the precursor protein across the membrane. (4) Polar and negatively charged residues introduced into the N-terminal region affect the secretion of the mature protein by preventing its correct folding.