Effect of dietary oligochitosan supplementation on ileal digestibility of nutrients and performance in broilers.

The effect of dietary chitosan oligosaccharides (COS) supplementation on ileal digestibilities of nutrients and performance in broilers was assessed by feeding graded levels (0, 50, 100, 150 mg/kg) of COS. Two thousand four hundred male commercial Avian broilers (1-d-old) were assigned randomly to 5 dietary treatment groups (60 birds per pen with 8 pens per treatment). Diet A was a typical corn- and soybean meal-based diet supplemented with 6 mg/kg of an antibiotic flavomycin (positive control). Diet B was the basal diet without any supplement. Diets C, D, and E were formulated by adding 50, 100, and 150 mg/kg of COS to the basal diet, respectively. On the morning of d 21 and 42, 64 birds (8 per pen with 8 pens per treatment) from the growth trial for each age group were killed by cervical dislocation for determination of the ileal digestibilities of nutrients. Dietary supplementation with COS and antibiotic enhanced (P < 0.05) the ileal digestibilities of DM, Ca, P, CP, and all amino acids (except for alanine in the 21-d-old birds or phenylalanine, glutamate, and glycine for the 42-d-old birds). Feed efficiency was improved (P < 0.05) in response to dietary supplementation of an antibiotic or COS (150 mg/kg for d 1 to 21, and 100 and 150 mg/kg for d 21 to 42). The results demonstrate for the first time to our knowledge that dietary COS supplementation was effective in increasing the ileal digestibilities of nutrients and feed efficiency in broilers. Our findings may explain a beneficial effect of COS on chicken growth performance.

Stachyose extract from Rehmannia glutinosa Libosch. to lower plasma glucose in normal and diabetic rats by oral administration.

The hypoglycemic effects of water extract and stachyose extract (Part III) from Rehmannia glutinosa Libosch. were investigated in this paper by oral administration to normal, glucose- and adrenaline-induced hyperglycemic and alloxan-induced diabetic rats. The results showed that Part III had the effect of lowering fasted plasma glucose level and partially preventing hyperglycemia induced by glucose (2.5 g x kg(-1), i.p.) and adrenaline (300 microg x kg(-1), i.p.), respectively, but no obvious dose-dependent effect was found when it was administered at the doses of 100, 200 and 400 mg x kg(-1) for 6 days, i.g. In alloxan-induced diabetic rats, Part III (200 mg x kg(-1) for 15 days, i.g.) gave a significant decrease in blood glucose level. The results suggested that Part III, which is mainly composed of stachyose from Rehmannia glutinosa Libosch., had a significant hypoglycemic effect in glucose- and adrenaline-induced hyperglycemic and alloxan-induced diabetic rats.

Aberrations of ammonia metabolism in ornithine carbamoyltransferase-deficient spf-ash mice and their prevention by treatment with urea cycle intermediate amino acids and an ornithine aminotransferase inactivator.

Sparse fur with abnormal skin and hair (spf-ash) mice are deficient in ornithine carbamoyltransferase (OCT) activity, but their OCT protein is kinetically normal. We administered ammonium chloride to spf-ash mice, in order to analyze ammonia metabolism and to find a rationale for the therapy of OCT deficiency. Ammonia concentration in the liver of spf-ash mice increased to a level much higher than in the control. Ammonium chloride injection caused an increase in ornithine (Orn) 5 min after injection and an increase in the sum of Orn, citrulline (Cit) and arginine (Arg) for at least 15 min in the liver of control mice, but no increase in Orn, Cit and Arg in the liver of spf-ash mice. Treatment of spf-ash mice with Arg 5-20 min prior to the injection of ammonium chloride kept the hepatic ammonia concentration at a level comparable to that without the load. A significant reciprocal relationship between ammonia and Orn concentrations in the liver of spf-ash mice 5 min after an ammonium chloride load with or without Arg strongly suggests that ammonia disposal is dependent on the supply of Orn. In spf-ash mice loaded with tryptone as a nitrogen source, Arg supplementation showed a dramatic decrease in urinary orotic acid excretion in a dose-dependent manner. Similar effects were observed with Cit and Orn at the same dose, and a long-lasting effect with an ornithine aminotransferase inactivator, 5-(fluoromethyl)ornithine, at a much lower dose. The rate of urea formation in liver perfused with ammonium chloride was lower in spf-ash mice than in controls, but with the addition of Orn to the medium it increased to a similar level in control and spf-ash mice. These results indicate that OCT is not saturated with Orn in vivo under physiological conditions and that the administration or enrichment of the urea cycle intermediate amino acids enhances the OCT reaction so that the ammonia metabolism of OCT-deficient spf-ash mice is at least partially normalized.

Retroviral transduction of human CD34+ umbilical cord blood progenitor cells with a mutated dihydrofolate reductase cDNA.

Umbilical cord blood cells (UCB) have become a major target population for experimental and clinical studies using transfer of genes involved in inborn enzymatic diseases. Cord blood contains hematopoietic progenitor cells at a high frequency, and expanding these cells ex vivo generates sufficient numbers of hematopoietic precursors for transplantation into adults, e.g., as supportive treatment. As clinical reports about retroviral transduction into UCB cells have not been as encouraging as the first preclinical data, we have established a retroviral transduction system that allows expansion and selection of hematopoietic progenitor cells from UCB. CD34-enriched UCB cells were transduced with a retroviral vector encoding a mutated dihydrofolate reductase cDNA that confers MTX resistance. We observed increased resistance to MTX in transduced granulocyte macrophage-colony forming units (CFU-GM) after co-culture of CD34+ UCB cells with the virus-producing cell line, or after incubation with virus-containing supernatant. The supernatant-based transduction protocol included a prestimulation with recombinant interleukin-1 (rhIL-1), rhkit-ligand, and rhIL-3 to increase the percentage of cells in S phase to greater than 50%. Using this protocol we measured a 72-fold expansion of CFU-GM and a 2.5-fold selective advantage of transduced versus nontransduced progenitor cells after exposure to low-dose methotrexate in liquid culture. Polymerase chain reaction analysis revealed integration of proviral DNA into the majority of transduced colonies before and after ex vivo expansion. The retroviral vector and transduction protocol reported here provides an experimental system for selection and expansion of retrovirally transduced progenitor/stem cells from UCB that may help improve the efficiency of current clinical gene therapy strategies.

Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction.

A double-copy Moloney leukemia virus-based retroviral construct containing both the NeoR gene and a mutant human dihydrofolate reductase (DHFR) cDNA (Ser31 mutant) was used to transduce NIH 3T3 and mouse bone marrow (BM) progenitor cells. This resulted in increased resistance of these cells to methotrexate (MTX). The transduced BM progenitor cells were returned to lethally irradiated mice. The recipients transplanted with marrow cells infected with the recombinant virus showed protection from lethal MTX toxicity as compared with mock-infected animals. Evidence for integration of the proviral DNA was obtained by amplification of proviral DNA by polymerase chain reaction (PCR) and Southern analysis. Sequencing a portion of the PCR-amplified human DHFR cDNA showed the presence of the mutation. These studies with the human Ser31 mutant DHFR cDNA gave results comparable with those obtained with the mutant murine DHFR cDNA (Leu to Arg22) in developing MTX-resistant BM. The Ser31 mutant human DHFR cDNA is currently being tested for infection of human CD34+ human BM and peripheral blood stem cells in vitro.

[Variations of the essential oils in Acorua calamus L. and their major compositions].

The variations of the essential oils in Acorus calamus and their major compositions with seasons and producing areas were investigated by steam distillation and internal standard method. The result has shown that June is the best season for A. calamus cropping and that the major components of the volatile oil in the same part of the plant from different producing areas have the same chemical structure type.

Long-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase.

A double-copy Moloney murine leukemia virus-based retroviral construct containing both the NEOr gene and a mutated dihydrofolate reductase cDNA (Leu 22-->Arg) was used to infect mouse bone marrow cells. The infected mouse marrow was returned to lethally irradiated mice. Primary, secondary, and even tertiary recipients transplanted with bone marrow cells infected with the recombinant virus showed protection from lethal methotrexate toxicity. The viral construct containing a SV-40 promoter in the U3 region of the 3' long terminal repeat appeared to be more effective than a similar construct containing the adenosine deaminase promoter, although both afforded protection. Evidence for integration into blood cells of both the NEOr gene and the mutated dihydrofolate reductase gene was obtained by polymerase chain reaction; sequencing of the amplified dihydrofolate reductase cDNA showed the presence of the point mutation. These results indicate that early hematopoietic progenitor cells in the mouse can be successfully transduced with a drug resistance gene.

Transfection with a cDNA encoding a Ser31 or Ser34 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance.

Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.

[Effects of Ziziphus spinosa Hu on serum lipoprotein and experimental atherosclerosis].

Rabbits of experimental group were fed synthetic food, cholesterol (0.4 g/day/rabbit) yolk powder (4 g/day/rabbit), and additionally pulp of Suanzao (10 g/kg/day). Three months later, compared with the results of the control group (TC 1334.8 +/- 327.8 mg; LDL 1261.9 +/- 356.6 mg and TG 270.8 +/- 66.9 mg), TC (574.6 +/- 271.8 mg), LDL (490.6 +/- 247.1 mg) and TG (89.7 +/- 7.8 mg) of the experimental group were significantly decreased, but in the experimental group HDL increased significantly (42.2 +/- 22.5 mg to 14.2 +/- 3.9 mg), and the AS degree of coronary artery was markedly reduced.