Single-locus control of saccharin intake in BXD/Ty recombinant inbred (RI) mice: some methodological implications for RI strain analysis.

The sac locus, with a major effect on saccharin preference, was discovered by Fuller (1974) in C57BL/6J (B6), DBA/2J (D2), and derived crosses, and is now supported in the BXD/Ty recombinant inbred (RI) series by a marked bimodal distribution in saccharin preference among 20 strains. The B6 allele led to increased saccharin preference compared to the D2 allele. Since the search for bimodal distributions reflecting major gene loci is an essential part of RI strain analysis, a new statistical method is proposed to test for bimodality, and comparisons are made to previously proposed methods. Another new RI method, quantitative trait loci (QTL) analysis, allows provisional detection and mapping of minor as well as major gene loci. Using this method as a screen, significant associations with saccharin preference were suggested with marker loci on portions of six chromosomes. One of these, the D12nyu1 locus on chromosome 12, was independently supported in a panel of standard (non-RI) inbred strains also tested for saccharin preference. It is unclear whether this reflects the sac locus.

Use of recombinant inbred strains to detect quantitative trait loci associated with behavior.

Recombinant inbred (RI) strains are valuable not only for detecting major gene segregation and linkage but also for identifying associations between behavior and quantitative trait loci (QTL) that account for relatively small amounts of variation in behaviors for which strain distribution patterns are not bimodal. When applied to published data on genetic markers and on behavior for BXD RI strains, the RI QTL association approach suggests the presence of QTLs on chromosomes 6 and 12 for open-field activity and on chromosomes 1, 2, and 17 for high-pressure seizure susceptibility. Because the RI QTL approach does not require that the progenitor inbred strains of a particular RI series differ, researchers could focus on the BXD RI series, for which the greatest number of genetic markers are available. Focusing on BXD would capitalize on the cumulative nature of RI research which permits analyses of QTL sources of genetic correlations across studies.

Use of recombinant inbred strains to identify quantitative trait loci in psychopharmacology.

Unlike simple Mendelian characteristics, individual differences in complex quantitative phenotypes studied in psychopharmacology are generally distributed continuously and are likely to be influenced by many genes. Recombinant inbred (RI) strains are valuable not only for their traditional use of detecting major gene segregation and linkage but also for identifying associations between quantitative traits and quantitative trait loci (QTL) that account for relatively small amounts of variation in phenotypes as well as loci that account for greater amounts of variation. When applied to published data on genetic markers and on amphetamine, alcohol, and morphine responses in BXD RI strains (RI strains developed from the cross between C57BL/6J and DBA/2J progenitor inbred strains), the RI QTL approach identified several significant associations beyond known major gene effects. Together, significant associations explain more than half of the genetic variance for these measures. The RI QTL approach is especially valuable for investigating the QTL underpinnings of genetic correlations among measures. It is recommended that psychopharmacogenetic research focus on the BXD RI strains. The cumulative and integrative nature of such a program of research is the major benefit of the RI QTL association approach for molecular genetic analysis of psychopharmacological processes, their physiological infrastructure, and their interface with other behavioral and biological systems.

Quantitative trait loci and psychopharmacology.

Unlike simple Mendelian characteristics, individual differences in behavior, including behavioral responses to drugs, are generally distributed continuously, show substantial non-genetic as well as genetic influence, and appear to be influenced by many genes rather than one or two major genes. For these reasons, application of techniques of molecular biology to identify DNA sequences responsible for behavioral variation requires strategies that can detect genes that account for small amounts of variation, so-called quantitative trait loci (QTL). One such strategy involves analyses of association using recombinant inbred (RI) strains of mice. The RI QTL approach is especially valuable when researchers use the same RI series, such as BXD, which has 26 strains and more than 300 mapped genetic markers. Even when the progenitor inbred strains do not differ and when the strain distribution pattern of the RI strains is continuous, the approach can be used to identify and map QTL and estimate the extent to which the QTL account for genetic variance for a particular phenotype. A multivariate extension of this approach can assess genetic correlations among measures as well as the QTL underpinnings of these genetic correlations. The cumulative and integrative nature of such a program of research is the major benefit of the RI QTL approach for molecular genetic analysis of psychopharmacological processes, their physiological infrastructure, and their interface with other biological and behavioural systems.

Quantitative trait loci and psychopharmacology: response to commentaries.

The theme of our article was that a merger is needed between quantitative genetic and molecular genetic approaches in order to detect genes associated with psychopharmacological processes even when the genes account for small amounts of variance, so-called quantitative trait loci (QTL). The recombinant inbred (RI) QTL approach using the BXD RI series was discussed as a promising approach.The commentaries by Crabbe and by McGuffin and Buckland make several excellent points to which we have little to add. Goldman and Katz, on the other hand, disagree with some of our arguments and for this reason much of the limited space of our response to the commentaries is directed towards the commentary by Goldman and Katz. We focus on three general issues that they raise: the relationship between quantitative genetics and molecular genetics (single genes vs multiple genes), reverse genetics (anonymous markers) vs forward genetics (candidate genes) and heterogeneity (narrow vs broad assessment). Our response to Goldman and Katz is that these are false dichotomies-we do not need to choose sides between major- and multiple- gene approaches, reverse and forward genetics, or narrow and broad assessment. Rather than choosing sides, we should encourage the deployment of multiple research strategies in order to maximize the probability of identifying genes that affect behavior. A major strength of the RI QTL approach is that it can identify both major- and multiple-gene effects, it employs both reverse and forward genetics, and it can be applied to both narrow and broad assessment (and its multivariate extension is ideally suited to understanding the genetic interrelationships among different levels of assessment).After discussing these general issues raised by Goldman and Katz, we address two specific issues raised in the commentaries: multivariate analysis of multiple markers and the use of F(1) crosses between RI strains to increase power. We end by mentioning the establishment of an RI QTL collaborative registry which aims to facilitate RI QTL analyses.

S1-hypersensitive sites in eukaryotic promoter regions.

We have examined by fine mapping the S1 nuclease-hypersensitivity of the 5' flanking regions of the human beta-globin and rat preproinsulin II genes and of the SV40 origin/enhancer region. In all cases S1-hypersensitive sites are located in known or presumed promoter/regulatory regions. Though a consensus DNA sequence is not evident, all of these sites reside in predominantly homopurine-homopyrimidine stretches. The alternate (non-B) DNA structure which is revealed by the enzymatic probe is a sequence-dependent feature of a short stretch of DNA, which is retained upon transplantation into a foreign environment. The alternate structure exhibits S1-nicking patterns uniquely different from those associated with the presence of Z-DNA.

The interaction of immobilized thrombin with human antithrombin III.

Interaction of human antithrombin III (AT III) with human alpha-thrombin coupled to Sepharose 4B was investigated. Despite markedly reduced esterolytic, amidolytic and especially coagulant activity, more than 90% of immobilized thrombin formed stable complexes with purified AT III. Presence of high affinity heparin did not facilitate the inhibition to the degree seen in reactions conducted with soluble thrombin. Instead, heparin induced proteolysis of up to 66% of the inhibitor that remained in solution. This led to the isolation of a homogeneous protein fragment which migrated in SDS-gel electrophoresis as a band of 50,000 Mr, cross-reacted with antibodies to human AT III but showed no biologic activity nor sufficient affinity for heparin. Out of the three major inhibitors capable of binding soluble thrombin in human plasma, only AT III reacted with immobilized thrombin. However, Sepharose-coupled thrombin mixed with plasma in the presence of heparin produced outstanding quantities of residual immunoreactive AT III devoid of inhibitory activity. These data suggest that presence of high affinity heparin in the environment of thrombin attached to a solid support may dramatically decrease the efficiency of enzyme inhibition.

High molecular weight forms of antithrombin III complexes in blood.

A double antibody competition radioimmunoassay was developed that allowed to detect specifically as little as 15 ng antithrombin III (AT III) per ml of the assayed material. In normal plasma examined by this assay, AT III concentration averaged 199 +/- 21 micrograms/ml. Complexes of AT III with thrombin or factor Xa crossreacted with free AT III in 87% and 95%, respectively. Molecular forms of AT III produced in plasma treated with coagulation enzymes, or in serum, were assessed by measuring immunoreactive AT III in fractions obtained by gel filtration chromatography on Sephadex G-200. AT III bound by thrombin in fibrinogen free-plasma ranged in molecular size from 160,000 to above 250,000. Similar aggregation occurred when monomeric complex of purified AT III and thrombin, of 90,000 Mr, was added to plasma. Presence of heparin intensified the degree of aggregation. In factor Xa-treated plasma AT III was converted into components with 160,000 Mr, or less. No complexes below 200,000 Mr were present in serum. They decreased in size to 160,000 Mr after affinity chromatography on heparin-Sepharose. These results indicated that blood represents a unique milieu conducive to aggregation of bound AT III. It appears, however, that AT II complexes present in blood may not only aggregate, but also associate with other serum proteins through unstable binding most likely caused by the enzyme component of the complex.

Enhancement by heparin of thrombin-induced antithrombin III proteolysis: its relation to the molecular weight and anticoagulant activity of heparin.

Previous findings indicated that binding of heparin to antithrombin III (AT III) facilitates thrombin-induced proteolysis of the inhibitor. We now studied this property of heparin in regard to its molecular weight and anticoagulant activity. Commercial heparin was resolved on Sephadex G-200 into six fractions of decreasing molecular weight. From each fraction high affinity (HA) heparin was isolated by chromatography on AT III-Sepharose and examined in reaction of alpha-thrombin with a molar excess of 125I AT III. Proteolysis of the inhibitor was assessed by SDS polyacrylamide gel electrophoresis. In the presence of the HA heparin from 18% to 38% of AT III participating in reaction appeared in the form of inactive 50,000-dalton fragment, as opposed to 7% of AT III fragmented in the absence of heparin. Although the ability to potentiate proteolysis was at its peak in the medium-molecular-size heparin fraction, the amount of degraded inhibitor relative to anticoagulant activity increased with decreasing molecular weight of the polysaccharide. These findings are consistent with the possibility that the ability of bound heparin to facilitate the cleavage of AT III by thrombin is generally less contingent upon secondary characteristics of the polysaccharide than the anticoagulant activity.

[Laboratory methods of examination of porous vascular prostheses]. / Laboratoryjne metody badania porowatych protez naczyniowych.

The author discusses applied methods of investigation of porous vascular prostheses with special attention to physical methods and proposes some heretofore unused criteria for the evaluation of polyester prostheses such as: quantitative determination of oligomers catalist residues, inor ganic fillers and manufacturer's oil and identification of the degree of crystallization and molecular orientation. The evaluation of these parameters in conjunction with the results of physical, mechanical, hematological, toxicologic and preclinical testing may form the basis for further improvement of vascular prostheses.