Potentiation of the D2 mutant motor phenotype in mice lacking dopamine D2 and D3 receptors.

Within the D2-class of dopamine receptors, the D2 and D3 subtypes share the highest degree of similarity in their primary structure. However, the extent to which these two receptor subtypes have similar or different functional properties is unclear. The present study used gene targeting to generate mice deficient for D2, D3, and D2/D3 receptors. A comparative analysis of D2 and D3 single mutants and D2/D3 double mutants revealed that D2/D3 double mutants develop motor phenotypes that, although qualitatively similar to those seen in D2 single mutants, are significantly more severe. Furthermore, increased levels of the dopamine metabolites dihydroxyphenyl acetic acid and homovanillic acid are found in the dorsal striatum of D2 single mutants. The levels of these metabolites, however, are significantly higher in mice lacking D2 and D3 receptors. In addition, results of immunoprecipitation experiments revealed that D2 single mutants express higher levels of D3 receptor proteins during later stages of their postnatal development. These results suggest that D3 receptors compensate for some of the lacking D2 receptor functions and that these functional properties of D3 receptors, detected in mice with a D2 mutant genetic background, remain masked when the abundant D2 receptor is expressed.

Artifactual strain-specific signs of incipient brain amyloidosis in APP transgenic mice.

In an attempt to generate transgenic mice modeling Alzheimer-type amyloidogenesis, the COOH-terminal 103 residue human APP segment was expressed in brain regions known to be vulnerable in AD. Transfected cells overexpressing this transgene were previously shown to develop intracytoplasmic inclusions that were immunoreactive with antibodies to the APP COOH-terminus. Transgenic C57B6/SJL mice produced transgene-coded mRNA in their brains at levels up to sixfold above endogenous APP, most abundantly within cortical and hippocampal pyramidal neurons. Immunocytochemistry with anti-A beta antibodies revealed occasional structures that resembled diffuse amyloid, but which could not be detected on serial sections. Immunolabeling with antibodies to APP regions NH2-terminal to the transgene-coded domain revealed elevated immunoreactivity within perikarya and neurites in regions expressing the highest transgene and endogenous APP mRNA levels, similar to observations previously reported within vulnerable neurons in AD brain. However, subsequent breeding revealed that this phenotype segregated with the B6/SJL background rather than the transgene, thus emphasizing the importance of genetic background to observations of putative AD-type pathology in transgenic animals.

The role of cholesteryl ester transfer protein in primate apolipoprotein A-I metabolism. Insights from studies with transgenic mice.

To assess the effects of cholesteryl ester transfer protein (CETP) on the primate lipoprotein profile, a transgenic mouse expressing cynomolgus monkey CETP was developed. The C57BL/6 mouse was used, and four lines expressing the primate CETP were established. The level of CETP activity in the plasma of the transgenic mice ranged from values similar to those obtained for the monkey to levels approximately sixfold higher than that in the normal monkey. When all of the lines were taken into consideration, there was a strong (r = -0.81 or higher, p less than 0.01) negative correlation between plasma CETP activity and total plasma cholesterol, plasma apolipoprotein (apo) A-I levels, and plasma apo A-I to apo B ratio. There was a strong positive correlation (r = 0.77) between plasma CETP activity and plasma apo B levels. The size of the apo A-I-containing lipoproteins was significantly reduced in mice with high plasma CETP activity, and that reduction in size was due to the absence of the larger (HDL1 and HDL2) apo A-I-containing particles in the plasma. When the transgenic mice were fed a high-fat, high-cholesterol diet, the effects of the diet on lipoprotein profile were more prominent in the CETP transgenic mice than the controls. The CETP transgenic mice had, for example, substantially higher plasma cholesterol and plasma apo B levels (p less than 0.01), and the apo B-containing lipoproteins were generally larger than those in the nontransgenic C57BL/6 mice consuming the same diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene.

The fibrinolytic potential of the vasculature is modulated primarily by the availability and activity of plasminogen activators, which convert the zymogen plasminogen into the active fibrin-degrading enzyme plasmin. The activities of these key regulatory enzymes are directly neutralized by their primary endogenous inhibitor, plasminogen activator inhibitor-1 (PAI-1). Although some individuals with a tendency to develop thrombotic disorders exhibit elevated levels of PAI-1 in their plasma, the cause-and-effect relationship between increased PAI-1 and thrombosis is still unclear. Specifically, it is not known whether chronic depression of fibrinolytic activity results in the development of thrombosis. To address this question we developed transgenic mice in which the contribution of PAI-1 to thrombus formation could be evaluated. The results presented in this report indicate that elevated levels of PAI-1 contribute to the development of venous but not arterial occlusions.

Apo A-I metabolism in cynomolgus monkeys: male-female differences.

Female cynomolgus monkeys have significantly higher plasma apo A-I concentrations than males (P = 0.04) and are able to maintain higher levels than the males even after consuming a high-cholesterol diet that severely depresses the apo A-I concentration in primates (P less than 0.05). The mechanism responsible for this difference was investigated by comparing apo A-I turnover (synthesis and catabolism) in males and females consuming monkey chow and in a separate group of males and females that had consumed the high-cholesterol diet for several weeks. The average length of time an apo A-I molecule remained in the plasma compartment of chow-fed monkeys was 2.62 days but decreased to 1.52 days (P less than 0.01) in animals fed the HC diet. There were no male-female differences in the residence times. The absolute turnover rate (mg/day) of apo A-I was not statistically affected by diet or sex; however, the females were substantially smaller than the males (3.8 vs. 4.8 kg; P less than 0.01) and their plasma volumes were significantly smaller than those of the males, even after correction for differences in body wt. (32.6 vs. 37.0 ml/kg, respectively; P less than 0.01). Taken together, the data indicate that females cynomolgus monkeys have higher apo A-I synthesis rates than males of comparable plasma volume (P = 0.03), which we would propose accounts for the higher plasma apo A-I concentrations evident in females.

Purification, cloning and nucleotide sequence determination of cynomolgus monkey apolipoprotein C-II: comparison to the human sequence.

We have purified apolipoprotein C-II (apo C-II) from cynomolgus monkey plasma, prepared antibody against it and used the antibody to isolate a cDNA containing the complete coding sequence for cynomolgus monkey apo C-II. Sequence analysis indicated that the monkey apo C-II cDNA was 200 bp longer than the human and the difference in size was all in the 5 degrees untranslated region of mRNA. This was confirmed by Northern analysis of human and monkey RNA. There was an open reading frame in the monkey apo C-II cDNA sequence encoding a preprotein of 101 amino acids - identical in size to the human protein. The carboxyl terminal 44 amino acids of the protein were 100% homologous to the human apo C-II amino acid sequence indicating evolutionary conservation of both structure and function. However, the amino terminal 35 amino acids of the protein were only 75% homologous and the amino terminal 19 amino acids were only 58% homologous to the human sequence. The amino acid sequence derived from the nucleotide sequence predicts a more basic protein than the human apo C-II and this is confirmed by isoelectric focusing and immunoblotting.

Molecular cloning and characterization of complementary deoxyribonucleic acids corresponding to bovine trophoblast protein-1: a comparison with ovine trophoblast protein-1 and bovine interferon-alpha II.

Bovine trophoblast protein-1 (bTP-1) is a secreted glycoprotein that consists of several forms differing slightly in mol wt and isoelectric point. It is produced by bovine conceptuses after about day 15 of pregnancy and is believed to play a key role in signalling the presence of an embryo to the mother. In this study, a series of recombinant cDNA clones corresponding to the mRNA for bTP-1 have been isolated from cDNA libraries representing day 18-19 bovine conceptus poly(A)+ mRNA. Base sequencing of several cDNAs indicated that multiple mRNAs for bTP-1 exist. Northern blotting and primer extension experiments showed that the mRNAs average about 1 kilobase in length. One apparently full-length cDNA clone consisted of 1035 bases up to the beginning of the poly(A) tail. It contained an open reading frame of 195 codons which began at a position 79 bases from the 5' end. Its entire sequence was 85% identical to that of a cDNA for the immunologically related ovine trophoblast protein-1 (oTP-1) and about 79% identical to that for a bovine interferon-alpha II (IFN alpha II). The highest conservation of sequence (greater than 90%) was noted in the 3'-untranslated sequences of the bTP-1 and oTP-1 cDNAs. The deduced amino acid sequence of bTP-1 shared 80% identity with oTP-1, between 45-55% with human, rodent, porcine, and bovine IFNs of the alpha 1 subfamily and about 70% with a bovine IFN alpha II. A single potential site for N-glycosylation was noted at Asn78. These results show that bTP-1, like its ovine counterpart oTP-1, is structurally related to the IFN alpha S. We suggest that these embryonic IFNs play a role in controlling immunoreactions at the trophoblast-uterus interface as well as triggering other maternal responses to pregnancy.

Interferon RNA of embryonic origin is expressed transiently during early pregnancy in the ewe.

Ovine trophoblast protein-1 (oTP-1), an interferon of embryonic origin, is produced during the peri-implantation period of early pregnancy. Secretion of oTP-1 is detectable between days 13 and 21, but not beyond. In this study, the levels of oTP-1 mRNA in embryos were analyzed to determine if they reflected the transient nature of oTP-1 production. Total cellular RNA (tcRNA) was isolated from embryos representing day 12 (n = 5), 14 (n = 7), 16 (n = 5), 18 (n = 6), 20 (n = 4), and 22 (n = 5) of pregnancy and spotted on nylon membranes. Complementary RNA was transcribed from a specific oTP-1 cDNA (550 base pairs) template and applied (16-1000 pg) to nylon membranes to develop a standard curve. The fixed RNA samples were then allowed to hybridize with the 32P-labeled oTP-1 cDNA. oTP-1 mRNA was not detectable on day 12, increased to high levels (3.6 +/- 1.6 ng/ug of embryo DNA) on day 14, decreased about 5-fold by day 16, 15-fold by day 18, 170-fold by day 20, and 200-fold by day 22 of pregnancy. At day 14 oTP-1 mRNA comprised 0.060 +/- 0.019% of the tcRNA and was more abundant than actin mRNA. Northern analyses of pooled tcRNA representing each day of pregnancy showed that the oTP-1 probe hybridized to a single class of mRNA (approximately 1.1 kilobases) and confirmed the results obtained with dot blots.

Interferon-like sequence of ovine trophoblast protein secreted by embryonic trophectoderm.

In most species the length of a pregnancy exceeds that of the luteal phase of the ovarian cycle. The conceptus within the uterus, therefore, is believed to produce a substance or substances which directly or indirectly prolong the lifespan of the corpus luteum and prevent a return to ovarian cyclicity. This phenomenon is known as maternal recognition of pregnancy. The active substance implicated in signalling maternal recognition of pregnancy in the sheep is an embryonic secretory protein, known as ovine trophoblast protein-1 (oTP-1) (refs 2-4), which is targeted in a paracrine manner to the uterine epithelium of the mother. We report here the primary amino-acid sequence of oTP-1 as inferred from a cloned complementary DNA and demonstrate that the protein is most probably an interferon-alpha.

The primary structure of cynomolgus monkey apolipoprotein A-1 deduced from the cDNA sequence: comparison to the human sequence.

We have cloned and analyzed a cDNA containing the complete coding sequence for cynomolgus monkey apolipoprotein A-1 (apoA-1). This cDNA clone was found to share approx. 97% nucleotide sequence identity with the published human apoA-1 and encodes a protein of the same size as the human protein. Paired proline residues are present at positions 3 and 4 in the mature protein as has been reported for other primate species and the propeptide sequence is identical to the human propeptide. The amino acid content derived from the nucleotide sequence predicts a more basic protein than human apoA-1 and this was confirmed by isoelectric focusing analysis. In addition, we present evidence for two different transcriptional initiation sites for the cynomolgus monkey gene in contrast to only one for human.