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Our previous study showed dietary supplementation with Arg and Glu increased intramuscular fat deposition and decreased back fat thickness in pigs, suggesting that the genes involved in lipid metabolism might be regulated differently in muscle and s.c. adipose (SA) tissues. Sixty Duroc × Large White × Landrace pigs with an average initial BW of 77.1 ± 1.3 kg were randomly assigned to 1 of 5 treatment groups (castrated male to female ratio = 1:1). Pigs in the control group were fed a basic diet, and those in experimental groups were fed the basic diet supplemented with 2.05% L-alanine (isonitrogenous group), 1.00% L-arginine (Arg group), 1.00% glutamic acid + 1.44% L-alanine (Glu group), or 1.00% L-arginine + 1.00% glutamic acid (Arg+Glu group). Fatty acid percentages and mRNA expression levels of the genes involved in lipid metabolism in muscle and SA tissues were examined. The percentages of C14:0 and C16:0 in the SA tissue of Glu group pigs and C14:0 in the longissimus dorsi (LD) muscle of Glu and Arg+Glu groups decreased (P< 0.05) compared to the basic diet group. The Arg+Glu group showed the highest (P< 0.05) hormone-sensitive lipase expression level in SA tissue and higher (P< 0.05) mRNA levels of PPARγin the LD muscle than the basic diet and isonitrogenous groups. Additionally, the mRNA level of fatty acid synthase in the Arg+Glu group was more upregulated (P< 0.05) than that of the Arg group. An increase in the mRNA level of PPARγin the biceps femoris muscle was also observed in the Arg+Glu group (P< 0.05) compared with the basic diet and isonitrogenous groups. Collectively, these findings suggest that dietary supplementation with Arg and Glu upregulates the expression of genes involved in adipogenesis in muscle tissues and lipolysis in SA tissues.

Sixty Duroc × Large White × Landrace pigs with an average initial BW of 77.1 ± 1.3 kg were used to investigate the effects of dietary supplementation with arginine and glutamic acid on growth performance, carcass traits, and meat quality in growing-finishing pigs. The animals were randomly assigned to 1 of 5 treatment groups (12 pigs/group, male:female ratio 1:1). The pigs in the control group were fed a basal diet (basal diet group), and those in the experimental groups were fed the basal diet supplemented with 2.05% l-alanine (isonitrogenous group), 1.0% l-arginine (Arg group), 1% glutamic acid + 1.44% l-alanine (Glu group), or 1.0% l-arginine + 1.0% glutamic acid (Arg+Glu group). After a 60-d period of supplementation, growth performance, carcass traits, and meat quality were evaluated. The results showed no significant differences (P> 0.05) in growth performance and carcass traits of the pigs in the Arg group relative to the basal diet group; however, the longissimus dorsi (LD) muscle and back fat showed a decrease (P< 0.05) in the percentage of SFA. In the Glu group, the final BW, phase 1 (d 1 to 30) and phase 2 (d 31 to 60) ADFI, and average back fat thickness of the pigs decreased (P< 0.05) by 7.14%, 23.43%, 8.03%, and 33.88%, respectively, when compared with the basal diet group. Dietary Arg+Glu supplementation had no effect (P> 0.05) on the final BW, phase 2 ADFI, and average daily weight gain in pigs but decreased (P< 0.05) their phase 1 ADFI, average back fat thickness, and percentage of SFA in the LD muscle and back fat, and increased (P< 0.05) the i.m. fat (IMF) content of the LD and biceps femoris muscles when compared with the basal diet group. Furthermore, a 16% decrease in yellowness (b* value; P< 0.05) was observed in the Arg+Glu group pigs when compared with the isonitrogenous group. These findings suggest that dietary supplementation with both Arg and Glu beneficially increases the IMF deposition and improves the meat color and fatty acid composition without affecting growth performance and s.c. fat in pigs, providing a novel strategy to enhance meat quality in growing-finishing pigs.

Polyamines are essential for embryonic and fetal survival, growth, and development. Additionally, polyamines may induce autophagy in mammalian cells. However, little is known about the availability of polyamines or autophagy in the porcine conceptus with intrauterine growth restriction (IUGR). The present study was performed to evaluate the developmental changes of polyamine concentrations in IUGR and normal porcine fetuses as well as autophagic marker levels in the fetal intestinal mucosa during the second half of gestation when most fetal growth occurs. Allantoic fluid (ALF), amniotic fluid (AMF), umbilical vein, and the small-intestinal mucosa were obtained from both IUGR and normal fetal pigs at d 60, 90, and 110 of gestation. Concentrations of polyamines in fetal fluids as well as protein abundances of microtubule-associated protein light chain 3B (LC3B), an autophagic marker, in the fetal small-intestinal mucosa were determined. Concentrations of polyamines varied greatly in different fetal compartments and changed substantially with advancing gestation. Concentrations of polyamines in IUGR fetal fluids and the small-intestinal mucosa were markedly different from those in their normal counterparts at d 60 and 90 of gestation, whereas most of the differences were not detected by late (d 110) gestation. Specifically, polyamine levels were lower in the umbilical vein plasma but higher in ALF and AMF from IUGR fetuses. Furthermore, enhanced levels of an autophagic marker were observed in the small-intestinal mucosa of IUGR fetuses throughout mid and late gestation in association with abnormal spermidine levels in fetal plasma. These findings support the notion that enhanced autophagy may be an important survival mechanism in IUGR fetuses. Collectively, our findings provide a new framework for future studies to define the roles for polyamines in the prevention and treatment of IUGR in both human medicine and animal production.

It is well known that β-conglycinin, a soybean allergen, induces allergies and causes intestinal damage in fetuses and neonates. However, the underlying mechanisms responsible for the adverse effects of β-conglycinin remain elusive. In particular, it is unknown whether or not this dietary substance causes direct damage affecting the proliferation and integrity of intestinal cells. This study evaluated the effect of different concentrations of β-conglycinin (0 to 1,500 µg/mL) and the duration of culture (48 or 72 h) on the proliferation and proteome of porcine intestinal epithelial cells. Eight individually housed piglets (10 d old; initial BW, 3.79 ± 0.07 kg) were randomly divided into 2 groups (n = 4) and challenged with or without β-conglycinin via oral administration d 10 through 28. After the last administration of β-conglycinin or PBS, piglets were killed and jejuna mucosal samples were collected for proteomic analysis. Supplementing β-conglycinin to either culture medium or weanling pigs increased (P< 0.05) the expression of proteins related to apoptosis, stress, and inflammation, but decreased (P< 0.05) the expression of proteins related to cytoskeleton and nucleus replication in intestinal cells. Further analysis confirmed an increase in caspase-3 expression in the cells exposed to β-conglycinin in vivo and in vitro. Collectively, these novel results indicate that β-conglycinin directly induces intestinal damage by depressing intestinal-cell growth, damaging the cytoskeleton, and causing apoptosis in the piglet intestine.

Piglets obtaining milk from anterior and middle mammary glands (MG) grow faster than those suckling posterior MG, but the underlying mechanisms are not clear. The purpose of this study was to investigate the differential proteomes of colostrum and milk secreted by anterior and posterior MG. Six healthy primiparous sows with 7 pairs of MG were used; the first and the second pairs were defined as anterior MG and the sixth and seventh pairs as posterior MG. Colostrum and milk were collected at d 1 and 14 after parturition, respectively. Comparative proteomics analysis was performed to identify the differentially expressed proteins in colostrum and milk secreted by anterior and posterior MG. Results show that protein composition in colostrum and milk varied markedly with the anatomical location of MG. Immunoglobulins, lactadherin, and haptoglobin were upregulated (P< 0.05) in colostrum from anterior MG compared with posterior MG. Concentrations of immunoglobulins and lactoferrin in milk from anterior MG were greater (P< 0.05) than milk from posterior MG. Moreover, concentration of proteins from somatic cells was greater (P< 0.05) in milk from posterior MG compared with anterior MG. Most proteins, in which abundance was upregulated in colostrum and milk from anterior MG, contribute to passive immunity, intestinal development of suckling piglets and epithelial integrity, and the health of MG. Collectively, these results indicate that in comparison with posterior MG, anterior MG are more active in protein synthesis and produce more immunoglobulins and lactoferrin in colostrum and milk.

A recombinant E. coli ribonuclease H (RNase H) was chemically coupled to an antisense oligodeoxynucleotide (ODN) against the 5‘-noncoding region (5‘-NCR) of the hepatitis C virus. Purity of the conjugates was confirmed by sodium deodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) as a band corresponding to approximately 23 kDa. Conjugate function was tested by the cleavage of a HCV RNA transcript including the 5‘-NCR and core region and showed HCV sequence-specific cleavage by the appearance of an expected ~1000 nt fragment of RNA. Cleavage was not seen by RNase H alone, or ODN alone. Delivery studies using ³²P- and ¹²⁵I-labeling showed that while RNAse H failed to enter cells, the conjugate was efficiently taken into the cells. To assess intracellular effects, a cell line, Huh-7/CMV−NCRCΔluc, which expresses HCV mRNA (nt 1−585) fused to a marker gene, was transfected with the conjugate. Reporter gene expression was suppressed by 51.2% with the conjugate compared to only 39.7% by ODN alone, 35.8% by a mixture of RNase H plus ODN, and not at all by RNase H alone. In conclusion, the RNase H−ODN conjugate effectively cleaved an HCV transcript in vitro and inhibited expression of an HCV-marker fusion construct in a liver-derived cell line.

The purpose of this study was to compare the efficacy and safety of risperidone and haloperidol in treatment-resistant chronic schizophrenic patients. Subjects (n= 78) who met DSM-III criteria for schizophrenia were randomly assigned to receive 6 mg/day of risperidone or 20 mg/day of haloperidol for 12 weeks. Clinical efficacy was determined using the Positive and Negative Syndrome Scale (PANSS), and side-effects with the Treatment Emergent Symptom Scale (TESS). Risperidone produced a mean 39.8 ± 24.1% reduction in total PANSS score compared to a mean 28.3 ± 19.4% reduction in the haloperidol group (P< 0.05). Analysis of changes for the three subscores of the PANSS revealed that the general psychopathology and negative subscores were significantly improved in the risperidone group compared to the haloperidol group. As for the side-effects, the risperidone group showed a significantly lower TESS total score, as well as nervous system symptoms subscore and cardiovascular symptoms subscore, compared to the haloperidol group. Risperidone appears to be a more effective and better tolerated antipsychotic drug in treatment-refractory Chinese schizophrenia than haloperidol.

Ribonuclease H (RNaseH) recognizes and efficiently cleaves the RNA strand of DNA−RNA hybrids, but has no inherent sequence selectivity. However, the formation of DNA−RNA hybrids does require specific sequence recognition. On the basis of this concept, we wondered whether antisense oligonucleotides complementary to target RNA covalently linked to RNase H could be used to direct specific cleavage events mediated by RNase H. The aim of this research was to couple a DNA oligonucleotide to RNase H to confer specificity of ribonuclease activity toward hepatitis B viral (HBV) mRNA. A modified 13-base oligonucleotide that is specific for the DR1 region of HBV mRNA was conjugated to modified E. coli RNase H using a water soluble cross-linker. A 1200 base fragment of HBV RNA including the DR1 region was synthesized as a substrate using T7 RNA polymerase. Incubation of the RNase H−oligonucleotide conjugate with the RNA transcript resulted in cleavage of the HBV mRNA transcript in a concentration dependent manner. Eighty-five percent of substrate was cleaved under optimal conditions. Controls consisting of RNase H alone, oligonucleotide alone, and incubation of the conjugate with an unrelated mRNA substrate resulted in no cleavage activity. RNase H coupled to an HBV antisense oligonucleotide can specifically cleave target HBV transcripts.

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid−polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10³ overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10¹−10² by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

β-Thalassemia is a common disease in Southern China and 10 different mutations or frameshifts are responsible for most types of β-thalassemia in this area. We studied 126 chromosomes of 80 β-thalassemia patients from the Guangxi, Guangdong, and Sichuan Provinces using the polymerase chain reaction followed by dot-blot hybridization with specific oligonucleotide probes. The most common mutation in the three provinces is the frameshift at codons 41-42, followed by the A→T mutation at codon 17. The A→G mutation at nt -29 of the promoter is common in Sichuan but not in the other two provinces. Three mutations (T→C at nt -30; G→T at IVS-I-1, and G→C at IVS-I-5) were not observed. These data were used to initiate a prenatal diagnosis program using the same techniques for identification. Eleven fetuses at risk for β-thalassemia have been diagnosed.

Summary Southwestern Yunnan, comprising the Yangtze and Shan-Thai microcontinents and the Simao block, has successively undergone subduction of an oceanic plate, followed by a collision of the microcontinents and intracontinental rifting associated with basaltic volcanism during Late Paleozoic to Mesozoic.

A new method for labeling oligonucleotides was developed to obtain high specific activity of radioactive probes. In an oligonucleotide molecule, two sequences were designed. One sequence, the 5', contains 19 nucleotides and serves as a template for probe synthesis. The second sequence, 3', contains a consensus sequence which forms a Pst I site after forming a complementary strand with the primer. In the presence of E. coli DNA polymerase I (Klenow fragment), α-32P dNTP and other dNTPs, a radioactive labeled oligonucleotide was synthesized by the primer extension method. After Pst I digestion, the probe was different from its template in length by 4 bp and could be separated from each other on urea-polyacrylamide gel electrophoresis (PAGE). A radioactive 01ionucleotide probe with extremely high specific activity up to 1010 dpm/μg could be obtained by the use of this method. The oligonucleotide probes have been used for the detection of the Hb E mutation in this report.

We present evidence that normal hepatocytes can be specifically protected from galactosamine toxicity in vitro by targeting an antagonist to these cells via receptor-mediated endocytosis. The strategy is based upon the following principles: 1) galactosamine is a highly selective hepatotoxin that causes a dose-dependent depletion of uridine intermediates; 2) galactosamine toxicity can be antagonized by supplemental administration of uridine; 3) normal hepatocytes possess unique cell-surface receptors that can internalize galactose terminal (asialo-)glycoproteins with subsequent degradation of the glycoprotein ligand. Based on these facts, we hypothesized that chemical coupling of a galactosamine antagonist to an asialoglycoprotein could result in cell-specific delivery and protection of normal hepatocytes by targeting the antagonist via asialoglycoprotein receptors. Using a model system consisting of freshly isolated rat hepatocytes (receptor (+)) and Morris 7777 rat hepatoma (receptor (-)) cells, sensitivity to galactosamine in vitro was determined and found to be similar for both types of cells. A targetable antagonist was synthesized by coupling uridine monophosphate to asialoorosomucoid in a molar ratio of 5 to 1. Exposure of Morris 7777 cells to the targetable antagonist in the presence of a toxic concentration of galactosamine did not protect these cells as evidenced by a steady decline in the number of viable cells in a fashion identical to cells treated with galactosamine alone. However, normal hepatocytes that received the conjugate in the presence of galactosamine were protected as their viable cell number remained the same as control (untreated) cells. Competition by an excess of asialoglycoprotein inhibited the protective effect of the conjugate, supporting the concept that the asialoglycoprotein component of the conjugate was responsible for the specific delivery of the antagonist to the target cells.

Familial hypercholesterolemia is an inherited disease in humans, caused by a deficiency of low density lipoprotein (LDL) receptors, that we have used as a model for developing liver-directed gene therapies. Our strategy is to reconstitute hepatic LDL receptor expression in vivo by administering a DNA-protein complex that is capable of targeting the delivery of functional LDL receptor genes to hepatocytes. Infusion of this DNA-protein complex into the peripheral circulation of a rabbit animal model for familial hypercholesterolemia resulted in hepatocyte-specific gene transfer and a temporary amelioration of hypercholesterolemia. This noninvasive approach to gene therapy should have applications in the treatment of a wide spectrum of human diseases.

The formation of the ternary molecular complex composed of α-methylstyrene (α-MS), ethyl acrylate(EA) and diethylaluminum chloride (AlEt2CI) in of CH2CI2 were examined by UV spectroscopy. The polymerization rate was first order in monomer concentration. The rate depends linearly on the square root of benzoyl peroxide(BPO) concentration and on the cube root of the AlEt2Cl concentration. These results can be explained by a mechanism involving the homopolymerization of a ternary molecular complex.

Antitumor immunotoxins were formed by covalently attaching the ribosome-inactivating protein ricin A chain (RA) to the antitumor antibodies BR96 and L6. In vitro cytotoxicity assays established that BR96-RA was cytotoxic to H2987 human lung adenocarcinoma cells (IC50 = 6 nM), while L6-RA exhibited very low levels of cytotoxic activity (18% cell kill at 67 nM). The virulence factor from the intracellular pathogen Listeria monocytogenes, listeriolysin O (LLO), was able to potentiate the cytotoxicity of BR96-RA and L6-RA by 120- and >1340-fold, respectively, resulting in IC50 values of approximately 50 pM. LLO also potentiated the cytotoxicity of the peptide anticancer drug bleomycin by a factor of >2500 but had no effect on the cytotoxic activities of the anticancer drugs cytarabine and etoposide phosphate. In addition, LLO did not potentiate the cytotoxic activity of unconjugated ricin A chain or L6-RA on H2987 cells that were saturated with L6 prior to conjugate treatment. These results are attributed to LLO-induced alteration of the intracellular trafficking of molecules that are incorporated into acidic vesicles.

A derivative of colchicine was synthesized, in a manner that preserved its important structural features, and conjugated to an asialoglycoprotein. The conjugate was characterized by ultraviolet−visible spectrophotometry and protein analysis. An average coupling ratio of 2 mol of colchicine per mole of asialoglycoprotein was achieved. The conjugate was stable to incubation in serum but was split into its separate components under chemically reducing conditions. Incubation with cells in culture revealed that the conjugate had antiproliferative activity similar to that of colchicine, but only in asialoglycoprotein receptor-containing cells. There was no effect at all on asialoglycoprotein receptor (−) cells. Furthermore, the antiproliferative effect of the conjugate on receptor (+) cells was blocked by addition of a large molar excess of free asialoglycoprotein. Immunofluorescence microscopy revealed disruption of microtubules in cell cultures that were pretreated with the conjugate. These results indicate that a colchicine conjugate that is taken up specifically into cells by asialoglycoprotein receptors and released intracellularly in a biologically active form can be prepared.

A soluble DNA carrier system was used to target a foreign gene specifically to liver in vivo via asialoglycoprotein receptors. The DNA carrier was prepared consisting of a galactose-terminal (asialo-)glycoprotein, asialoorosomucoid (AsOR), covalently linked to poly-L-lysine. The conjugate was complexed in a 2:1 molar ratio (based on AsOR content of the conjugate) to the plasmid, pSV2 CAT, containing the gene for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Intravenous injection of [32P]plasmid DNA complexed to the carrier demonstrated specific hepatic targeting with 85% of the injected counts taken up by the liver in 10 min compared to only 17% of the counts when the same amount of [32P]DNA alone was injected under identical conditions. Targeted pSV2 CAT DNA was detected at a level of 1.0 ng/g liver by hybridization of a [32P]pSV2 CAT cDNA probe to rat liver DNA extracted 24 h after intravenous injection of AsOR-poly-L-lysine-DNA complex containing 1.0 mg of DNA. Homogenates of livers taken 24 h after injection of the complex revealed that the targeted CAT gene was functional as reflected by the detection of CAT activity (approximately 4 microunits/mg protein). Livers from control animals that received individual constituents of the complex produced no CAT activity. Simultaneous injection of excess AsOR to compete with the AsOR-poly-L-lysine-DNA complex for uptake by the liver inhibited CAT gene expression. Assays for CAT activity in other organs (spleen, kidney, lungs) failed to demonstrate any activity in these organs. This new soluble DNA carrier system can permit targeted delivery of foreign genes specifically to liver with resultant foreign gene expression in vivo.

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.

The effects of ketone bodies on the metabolism of alanine and glutamine were studied in isolated extensor digitorum communis (EDC) muscles from 24 h-fasted chicks. (1) Acetoacetate and DL-beta-hydroxybutyrate (4 mM) markedly inhibit branched-chain amino acid (BCAA) transamination and alanine formation. (2) Ketone bodies (1 and 4 mM) increase the intracellular concentration and release of glutamate and glutamine, suggesting that inhibition of BCAA transamination does not limit intracellular availability of glutamate for alanine synthesis. (3) Ketone bodies (1 and 4 mM) do not affect glucose uptake by muscles, but decrease the rate of glycolysis as well as the intracellular concentration and release of pyruvate in muscles. (4) Addition of 12 mM-glucose increases the formation of alanine in muscles incubated in the absence of ketone bodies, but has no effect in muscles incubated in the presence of 4 mM ketone bodies. (5) Addition of 5 mM-pyruvate to the media prevents the inhibiting effect of ketone bodies on BCAA transamination and alanine synthesis. These results suggest that ketone bodies decrease alanine synthesis by limiting the intracellular availability of pyruvate, owing to inhibition of glycolysis, and inhibit BCAA transamination by decreasing the intracellular concentration of amino-group acceptors such as pyruvate in EDC muscles from fasted chicks.

This paper examines the copolymerzation of methyl acrylate (MA) and isobutylene (IB) with a complexed initiating system of AlEtCl2 and benzoyl peroxide (BPO). The effects of monomer ratio, monomer concentration, initiator composition and polymerization conditions on copolymer composition, degree of alternation, intrinsic viscosity and conversion were studied. An alternating copolymer was obtained when the [IB] / [MA] mole ratio was equal or greater than one.

DNA delivered to the liver by asialoglycoprotein receptor-mediated endocytosis is degraded in lysosomes within 48 h. To test the hypothesis that microtubular disruption should promote transgene persistence by interrupting endosomal translocation to lysosomes, plasmids containing bacterial chloramphenicol acetyltransferase (pSV2-CAT) or human bilirubin-UDP-glucuronosyltransferase-1 (pSVK3-hBUGT1) genes were complexed with asialoglycoprotein-polylysine conjugates, and 1 mg of the complexed DNA was injected intravenously into bilirubin-UDP-glucuronosyltransferase-deficient Gunn rats. 30 min before DNA injection, one group received 0.75 mg of colchicine/kg of body weight intraperitoneally, which was shown by immunofluorescent confocal microscopy to disrupt the microtubular network. Control rats received normal saline. In colchicine-pretreated rats receiving pSV2-CAT, hepatic chloramphenicol acetyltransferase activity persisted for 9-14 weeks, whereas in the saline-pretreated group the activity was detectable for 48 h only. In colchicine-pretreated Gunn rats receiving pSVK3-hBUGT1, the DNA persisted in liver for 10 weeks, bilirubin glucuronides were excreted in bile, and serum bilirubin levels declined by 25-35% in 2-4 weeks and remained reduced for 8 weeks. Without colchicine pretreatment, the DNA was detectable in liver for 2 days only, and serum bilirubin levels were not reduced. Thus, microtubular disruption provides a noninvasive method for prolonging the effect of liver-targeted gene therapy.

A new method is discussed which examines trace, dried bloodstains by gel in situ hybridization using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe to determine the sex of the bloodstain for forensic medicine application. The complete DNA is transferred directly by electrophoresis onto the gel intact, bypassing the possibilities of impurities contaminating the sample and of DNA degradation. The method has proven accurate for small (2.5-mm-diameter) samples aged up to eight years and is quick, simple, and easily read.

Methionine transamination is extensive in rat and chick skeletal-muscle homogenates, but is barely detectable in intact rat, but not chick, skeletal muscles. Branched-chain amino acids essentially block methionine transamination in intact muscles and homogenates from both species. The physiological significance of methionine transamination in skeletal muscle is questioned.

We present evidence that a foreign gene driven by natural mammalian regulatory elements can be targeted to hepatocytes and the resultant gene expression made to persist. This was accomplished using a soluble DNA carrier system consisting of two covalently linked components: 1) a polycation, poly-L-lysine, that can bind DNA in a strong but non-damaging interaction, and 2) an asialoglycoprotein which can be targeted specifically to hepatocytes by cell surface asialoglycoprotein receptors unique to this cell type.

We utilized the presence of hepatic receptors for galactose-terminal glycoproteins to achieve specific rescue of differentiated hepatocytes from methotrexate toxicity. This was accomplished by administration of a conjugate formed by covalent coupling of the methotrexate antagonist, folinic acid (leucovorin), to galactose-terminating (desialylated) bovine fetuin. Two cultured hepatocyte-derived cell lines were chosen to test rescue from methotrexate toxicity: Hep G2 cells are capable of receptor-mediated endocytosis of galactose-terminating glycoproteins; PLC/PRF/5 cells are not. In the presence of methotrexate alone, both cells lines were reduced in number to 20% of control populations by day 5. Growth rates of both lines returned to normal when free folinic acid was added to the medium containing methotrexate. When asialofetuin-folinic acid conjugate was given to both cell lines in the presence of methotrexate, Hep G2 cells doubled their numbers by day 4 and reached control (without methotrexate) populations by day 8. However, PLC/PRF/5 cells failed to respond to administered asialofetuin-folinic acid conjugate under identical conditions, and the growth curve was the same as that for cells which received methotrexate alone. Conjugates of asialofetuin bound to folic acid (folate derivative requiring dihydrofolate reductase activity for conversion to an active metabolite) and fetuin (non-galactose-terminal glycoprotein) linked to folinic acid also were made for control studies. These conjugates failed to rescue either cell line.

We present, here, evidence for a pretranslational role of procollagen propeptides in the regulation of collagen synthesis. Amino- and carboxyl-terminal type I procollagen propeptides were isolated and purified from chick calvaria and tendon cultures. Human lung fibroblasts (IMR-90) were incubated in medium containing varying concentrations of propeptides. Amino-propeptides at 10 nM caused an 80% decrease in collagen synthesis compared to control. Higher concentrations of amino-propeptides did not decrease collagen synthesis further and no significant effect on non-collagen synthesis was found throughout the entire concentration range. Carboxyl-propeptides also inhibited collagen synthesis. At 10 nM, collagen synthesis was decreased by 30% and a concentration of 40 nM caused an 80% reduction. However, at the latter concentration non-collagen synthesis was also affected, decreasing by 20% relative to control. To assess possible pretranslational effects of propeptides, IMR-90 fibroblasts were treated with varying concentrations of each propeptide and levels of type I procollagen mRNA was determined by dot hybridization with a 32P-alpha 2(I) cDNA probe. Both propeptides caused significant concentration-dependent decreases in procollagen type I mRNA levels. At 10 nM, the amino-propeptide resulted in a 55% decrease in collagen mRNA levels while at 40 nM these levels decreased by 72% compared to control. Carboxyl-propeptides were also inhibitory, decreasing mRNA levels by 33% at 10 nM and 73% at 40 nM. Messenger RNA levels of a representative noncollagenous protein, beta-actin, were unaffected by either propeptide throughout the concentration range.