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Maple syrup urine disease (MSUD) is a genetic disease caused by a deficiency of branched-chain keto acid dehydrogenase, a mitochondrial multienzyme complex responsible for the decarboxylation of leucine, isoleucine and valine. The complex consists of three subunits (E1, E2, and E3) and mutations in any subunit result in MSUD. No satisfactory treatment for MSUD is currently available. Here we report the successful use of retroviral gene transfer to restore leucine decarboxylation activity in fibroblasts derived from a MSUD patient containing a mutation in the E2 subunit. A full-length human E2 cDNA was inserted into a retroviral vector (MFG) and a stable CRIP producer line was generated. The amphotropic virus was then used to transduce mutant human fibroblasts. In untransduced mutant cells, 1-14C leucine decarboxylation activity was less than 2% that of the wild-type cells. Decarboxylation of 1-14C leucine in transduced mutant cells was restored to 93% of the wild-type level. Correct targeting of the expressed wild-type E2 protein to mitochondria was demonstrated by comparing the immunofluorescent pattern of E2 and a mitochondrial marker protein. Stable expression of enzyme activity has been obtained for at least 7 weeks. In contrast to most previous gene therapy attempts, which replace a single enzyme defect, the present results demonstrate complementation of a phenotype resulting from a gene defect whose product is a part of a multienzyme complex. Based on these results, studies can now be undertaken to investigate the feasibility of gene therapy to correct MSUD.

In order to gain better understanding of the function of hepatic lipase (HL) in vivo, we have generated mice that lack HL using gene targeting in embryonic stem cells. No mRNA for HL was detected in the liver of homozygous mutants, and no HL activity was detected in their plasma. Total cholesterol levels in plasma of mutant mice were increased by about 30% compared with wild type animals. Plasma phospholipids and high density lipoprotein (HDL) cholesterol were also increased, but plasma levels of triglycerides were not altered. Analysis of density fractions of plasma lipoproteins revealed that HDL1 (d = 1.02-1.04) was increased in homozygous mutants fed regular chow. In response to a diet containing high fat and high cholesterol, HDL cholesterol was doubled in the mutants, but was slightly decreased in the wild type mice. These results clearly demonstrate the importance of HL in HDL remodeling and metabolism in vivo. Various earlier studies suggested a role of HL in metabolism of triglyceride-rich particles, but the mutant mice appear to have no impairment in clearing them; the mutants clear exogenously introduced chylomicrons from plasma at a normal rate, and they tolerate acute fat loading as well as normal animals unless the loading is extreme. These differences may reflect species differences. However, it is also possible that the consequence of absence of HL as in our mutants is different from the consequence when nonfunctional HL protein is present as in the human HL-deficient patients and in rats treated with HL antibodies. We hypothesize that absence of HL in mutant mice allows other lipases to bind to the sites in the liver normally occupied by HL and facilitate the clearance of triglyceride-rich particles in these mice.

Apolipoprotein B (apoB) is a key structural component of several lipoproteins. These lipoproteins transport cholesterol, lipids, and vitamin E in the circulation. Humans that produce truncated forms of apoB have low plasma concentrations of apoB, beta-lipoproteins, cholesterol, and often vitamin E. This condition has been modeled in mice by targeted modification of the apoB gene. Homozygous transgenic mice display all of the hallmarks of the human disorder. Unexpectedly, approximately 30% of the perinatal homozygotes are exencephalic and of those that have closed neural tubes, approximately 30% are hydrocephalic. The latter condition has also been noted in a relatively small proportion of the heterozygous mice. Vital staining of gestational day 9 (GD9) homozygous offspring has illustrated a striking pattern of excessive cell death involving the alar plate of the hindbrain. Histological and scanning electron microscopic analyses have confirmed this finding. We speculate that varying degrees of affect, as noted among GD 9 and 10 embryos, lead to the spectrum of malformations, including hydrocephaly, present in term fetuses. Analysis of vitamin E deficiency as a possible causative factor has illustrated that homozygous fetuses, indeed, show this deficiency. Amelioration of the defects through alpha-tocopherol supplementation of the maternal diet has been explored. Further analyses of this transgenic mutant promise to provide significant information relative to the role of deficiency of vitamin E and other apoB dependent compounds in dysmorphogenesis.

Familial hypobetalipoproteinemia is an autosomal codominant disorder resulting in a dramatic reduction in plasma concentrations of apolipoprotein (apo) B, cholesterol, and beta-migrating lipoproteins. A benefit of hypobetalipoproteinemia is that mildly affected individuals may be protected from coronary vascular disease. We have used gene targeting to generate mice with a modified Apob allele. Mice containing this allele display all of the hallmarks of human hypobetalipoproteinemia: they produce a truncated apoB protein, apoB70, and have markedly decreased plasma concentrations of apoB, beta-lipoproteins, and total cholesterol. In addition, the mice manifest several characteristics that are occasionally observed in human hypobetalipoproteinemia, including reduced plasma triglyceride concentrations, fasting chylomicronemia, and reduced high density lipoprotein cholesterol. An unexpected finding is that the modified Apob allele is strongly associated with exencephalus and hydrocephalus. These mice should help increase our understanding of hypobetalipoproteinemia, atherogenesis, and the etiology of exencephalus and hydrocephalus.

Four ewes were utilized to determine the effects of prostaglandin (PG) F2 alpha, PGE2 and luteinizing hormone (LH) on activity of phospholipase C (PLC) in ovine luteal tissue. Corpora lutea were collected on d 10 post-estrus and six slices from one corpus luteum from each ewe were pre-incubated with [3H]-inositol prior to incubation with one of 6 treatments. Treatments were 1) control, 2) PGF2 alpha (100 ng/ml), 3) PGE2 (10 ng/ml), 4) LH (10 ng/ml), 5) PGF2 alpha + PGE2 and 6) PGF2 alpha + LH. Phospholipase C was determined indirectly by measuring the accumulation of [3H]-inositol mono-, bis- and tris-phosphates (IP, IP2, IP3). Effects of PGF2 alpha (0 vs. PGF2 alpha) and luteotropic treatment (0 vs. PGE2 vs. LH) and their interactions were determined by analysis of variance. There was a significant main effect of PGF2 alpha (P less than 0.01) as concentrations of IP, IP2, IP3 and total [3H]-inositol phosphates were greater in tissue slices treated with PGF2 alpha, regardless of luteotropic treatment. Within groups receiving no PGF2 alpha (1,3,4), no effect of luteotropic treatment was observed. Within groups receiving PGF2 alpha (2,5,6), LH caused a significant (P less than .05) increase in the accumulation of total [3H]-inositol phosphates. Thus, PGF2 alpha can stimulate the activity of PLC in ovine luteal tissue and LH can potentiate this effect.

Transgenic mice were used in an experiment that was designed to serve as a model of a possible approach to reducing the amount of carcass fat in meat animals. The objective was to reduce the number of adipocytes in transgenic mice thereby restricting the capacity to accumulate lipid. Our approach employed the technique of genetic ablation. The promoter for the adipocyte lipid binding protein gene was used in an attempt to direct expression of diphtheria toxin genes specifically to adipocytes. Three diphtheria toxin genes were used; they encode, respectively, an extremely cytotoxic wild type toxin, a less toxic attenuated toxin, and a nonfunctional toxin. While it was not possible to accurately assess effects of the transgenes on lipid accumulation, several informative observations were noted. A large percentage of transgenic founder mice that harbor either wild type or attenuated toxin genes are morphologically abnormal, die as neonates, or exhibit reproductive abnormalities including sterility or failure to transmit the transgene to offspring. In contrast, mice that harbor the nonfunctional toxin gene or are nontransgenic rarely have these same abnormalities. These results suggest that the transgenic mice are expressing the transgenes in cells other than adipocytes and that the aberrant production of functional toxin is responsible for the congenital abnormalities. The production of morphological and reproductive abnormalities in transgenic animals should be useful for investigating normal developmental processes.

This study examined the viability of pig oocytes at the germinal vesicle stage following cooling or cryopreservation. Cumulus-intact oocytes (n = 641) were collected from slaughterhouse pig ovaries and used in two experiments. In Exp. I the viability of 1) control, 2) cryoprotectant control (CC, 1.5 M glycerol/.5 M sucrose), 3) cooled (0 degrees C) and 4) cryopreserved (-196 degrees C) oocytes was assessed after no incubation or a 24-h incubation. Survivability was judged by morphological appearance, trypan blue exclusion and fluorescein diacetate staining. Survival rate of control oocytes (90%; based primarily on morphological appearance of the cumulus) incubated 0 h was greater (P less than .05) than that of all other groups, whereas survival rate of -196 degrees C oocytes (57%) was less (P less than .05) than that of all other groups. However, vital staining of 0 degrees C and -196 degrees C oocytes showed 0% survival rate as evidenced by trypan blue uptake and lack of fluorescence. The cumulus cells surrounding oocytes that were stored at 0 degrees C or -196 degrees C survived freezing as evidenced by trypan blue exclusion and intense fluorescence. Similar differences among treatment groups were found for oocytes incubated 24 h. Exp. 2 examined the temperature at which oocytes became sensitive to cooling. Oocyte death occurred when oocytes were cooled to 15 degrees C or lower. These results demonstrate that pig oocytes at the germinal vesicle stage did not survive cooling to 15 degrees C or below. When assessing the viability of cryopreserved cumulus enclosed oocytes it is important to use vital stains in conjunction with morphological appearance.

Two experiments were conducted to determine if the ability of oxytocin to stimulate release of prostaglandin (PG)F2 alpha from ovine uterine tissue involved activation of phospholipase C (PLC). In the first experiment, 9 ewes were injected with progesterone for 11 d (12 mg/d, im). On days 11 and 12, ewes received an injection of estradiol (100 micrograms, im). Caruncular endometrial tissue was collected on d 13 and incubated in the presence or absence of oxytocin (10(-6) M). Concentrations of PGF2 alpha and its metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), in culture media were determined by radioimmunoassay. PLC activity was determined by measuring the intracellular accumulation of 3H-inositol phosphates after preincubation with 3H-inositol. Concentrations of PGF2 alpha and total PGF (PGF2 alpha + PGFM) in culture media were greater for explants treated with oxytocin than for controls (p. less than .02, p less than .06, respectively). A similar effect of oxytocin on intracellular concentrations of 3H-inositol phosphates was observed (p less than .01). A second experiment was conducted to determine if agonists of second messengers, produced by activation of PLC, could stimulate release of PGF2 alpha from ovine endometrial tissue. Seven ewes were treated with progesterone and estradiol as in experiment 1. Explants of caruncular tissue from each ewe were incubated with 1) control medium, 2) A23187 (10(-5) M), 3) oxytocin (10(-6) M), 4) phorbol 12-myristate 13-acetate (PMA, 10(-7) M), 5) PMA + A23187 and 6) PMA + oxytocin. Significant stimulatory effects of oxytocin, PMA and A23187 on concentrations of PGF2 alpha and total PGF in culture media were observed (p. less than .05, p less than .1, p less than .1, respectively). In conclusion, oxytocin stimulated release of PGF2 alpha and activity of PLC in explants of ovine endometrial tissue in vitro. Second messengers associated with activation of PLC enhanced release of PGF2 alpha from ovine endometrial tissue.

Twenty ovariectomized ewes were used in an experiment designed to examine the interaction of progesterone, estradiol, and oxytocin in the regulation of uterine secretion of prostaglandin F2 alpha (PGF2 alpha). All ewes underwent a steroid pretreatment that mimicked the changes in progesterone and estradiol which occur during the six days immediately prior to estrus. After pretreatment, ewes were randomly assigned to 1 of 4 treatment groups: 1) control (n = 4); 2) estradiol-17 beta (n = 6); 3) progesterone (n = 4); and 4) progesterone and estradiol-17 beta (n = 6). Progesterone was injected twice daily for 15 days. The dose of progesterone varied with day postestrus in a manner designed to simulate endogenous luteal secretion of progesterone. Estradiol-17 beta was administered in s.c. Silastic implants. The implants maintained circulating concentrations of estradiol at 3 pg/ml. On Days 5, 10, and 15 of treatment, ewes were injected with oxytocin (10 IU in 1.0 ml saline, i.v.). Jugular venous blood samples were collected beginning one-half hour prior to and continuing for 2 hours post-oxytocin injection for quantification of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM). No changes in concentration of PGFM following injection of oxytocin were observed on Day 5 or 10 in any treatment group. Concentrations of PGFM increased following injection of oxytocin on Day 15 only in groups receiving progesterone. Both the area under the PGFM response curve (p = 0.08) and peak response (p = 0.06) were greater in ewes treated with progesterone and estradiol-17 beta than in those receiving progesterone alone.(ABSTRACT TRUNCATED AT 250 WORDS)