Hypoxia stimulates human preproendothelin-1 promoter activity in transgenic mice.

Significant elevations in endothelin (ET)-1 levels accompany many diseases, but the underlying regulatory mechanisms are unclear. To investigate the in vivo regulation of human preproendothelin-1 (PPET-1), we examined the activity of the PPET-1 promoter in transgenic mice exposed to hypoxia. Mice expressing one of three PPET-1 promoter-luciferase (PPET-1/LUC) reporter transgenes (approximately 2.5 kb, 138 bp, or none of the 5'-flanking sequences of the PPET-1 gene) were generated. LUC expression was reduced in mice with a truncated 138-bp PPET-1 promoter. Exposure of mice bearing the 2.5-kb PPET-1/LUC transgene to hypoxia (10% O2 for 24 h) increased LUC expression sixfold in pulmonary tissue but only twofold in other tissues. In situ hybridization revealed the strongest transgene expression in the pulmonary vasculature and bronchiolar epithelium. These data are consistent with the hypothesis that hypoxic induction of the PPET-1 gene leads to increased pulmonary production of ET-1 in diseases associated with low O2 tension.

Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1.

Transgenic mice overexpressing the chemokine monocyte chemoattractant protein-1 (MCP-1) in the thymus and central nervous system have a higher number of mononuclear cells in those tissues than do control littermates. In the thymus, there is a modest increase in the number of Mac-1 and F4/80 positive cells, but no apparent change in the number of lymphoid cells. A more pronounced mononuclear infiltrate is detected in transgenic mice expressing MCP-1 in the brain. The vast majority of the recruited cells in the brain are monocytes and macrophages, as defined by light microscopy, and ultrastructural and immunohistochemical criteria. Such cells are found in a perivascular orientation with minimal parenchymal infiltration, possibly as a consequence of the accumulation of MCP-1 in the vessels, as shown by immunohistochemistry. The mononuclear cell infiltrate in the brain can be significantly amplified by LPS treatment, suggesting that the recruitment properties of MCP-1 can be potentiated by additional factors.

Tissue distribution of amphotericin B lipid complex in laboratory animals.

Amphotericin B lipid complex (ABLC), under development for the treatment of serious fungal disease, is not a true liposome but a complex of amphotericin B, dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol with a particle size range of 1.6-6.0 microns. Tissue distribution of ABLC was determined in mice and rats after i.v. or i.p. administration. ABLC resembles typical liposomal preparations with amphotericin B concentrating in the reticuloendothelial system. After a single i.v. treatment with ABLC, amphotericin B was present in high concentrations in liver, lung and spleen of mice and rats while plasma levels were consistently low. Mouse liver contained 48% of the administered dose 1 h after treatment and always contained the largest amount of amphotericin B after ABLC treatment. In mice treated once daily for 7 consecutive days with 10 mg kg-1 ABLC, liver amphotericin B concentration reached 377 micrograms g-1. Tissue concentrations of amphotericin B were substantially lower when ABLC was given i.p. instead of i.v. with reticuloendothelial tissues containing 2- to 7-fold more after i.v. treatment. Animals treated with 10 mg kg-1 ABLC for 14 consecutive days showed no overt signs of toxicity and had only transient changes in liver and kidney function after treatment.

Evaluation of SQ 34,514: pharmacokinetics and efficacy in experimental herpesvirus infections in mice.

The new antiviral nucleoside SQ 34,514 [(1R-1 alpha, 2 beta, 3 alpha)-2-amino-9- [2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one], the active R isomer of racemic SQ 33,054 (cyclobut-G), was evaluated for efficacy in the treatment of herpesvirus infections in mice. SQ 34,514 was orally efficacious in a herpes simplex virus type 1 (HSV-1) systemic infection, an intracerebral HSV-2 infection, a vaginally induced HSV-2 infection in ovariectomized mice, and in a systemic murine cytomegalovirus infection. SQ 34,514 compared favorably with acyclovir and ganciclovir in the treatment of these experimental infections. In mice, SQ 34,514 had an oral bioavailability of 80% based on urinary excretion. SQ 34,514 may have potential value in the therapy of HSV and cytomegalovirus infections in humans.

Amphotericin B lipid complex therapy of experimental fungal infections in mice.

The amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, was evaluated for its acute toxicity in mice and for its efficacy in mice infected with a variety of fungal pathogens. ABLC was markedly less toxic to mice when it was administered intravenously; it had a 50% lethal dose of greater than 40 mg/kg compared with a 50% lethal dose of 3 mg/kg for Fungizone, the desoxycholate form of amphotericin B. ABLC was efficacious against systemic infections in mice caused by Candida albicans, Candida species other than C. albicans, Cryptococcus neoformans, and Histoplasma capsulatum. ABLC was also efficacious in immunocompromised animals infected with C. albicans, Aspergillus fumigatus, and H. capsulatum. Against some infections, the efficacy of ABLC was comparable to that of Fungizone, while against other infections Fungizone was two- to fourfold more effective than ABLC. Against several infections. Fungizone could not be given at therapeutic levels because of intravenous toxicity. ABLC, with its reduced toxicity, could be administered at drug levels capable of giving a therapeutic response. ABLC should be of value in the treatment of severe fungal infections in humans.

Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells.

1. Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined. 2. Incorporation of [35S]methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide. 3. Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides.

Identification of cDNAs corresponding to mosquito ribosomal protein genes.

Sequences encoding mosquito (Aedes albopictus) ribosomal proteins L8, L14 and L31 were identified from a cDNA library made from size-selected polyadenylated mRNA. Candidate cDNAs corresponding to moderately abundant mRNAs were screened by translation of hybrid-selected transcripts in wheat-germ lysates. Translation products were extracted with acetic acid and analyzed by electrophoresis in two dimensions in the presence of unlabeled ribosomal proteins. The identity of translation products that coelectrophoresed with purified ribosomal protein standards was supported by peptide mapping. The cDNAs corresponding to L8 (pL8) and L31 (pL31) hybridized to cytoplasmic mRNAs of 1.4 and 0.9 kb, respectively. In Southern blots of genomic DNA digested with BamHI, HindIII or EcoRI, the cDNA inserts from both pL8 and pL31 gave simple hybridization patterns suggestive of a low copy number for mosquito ribosomal protein genes.

Ceruloplasmin and metallothionein induction by zinc and 13-cis-retinoic acid in rats with adjuvant inflammation.

The induction of ceruloplasmin and metallothionein was investigated in rats with the early inflammatory phase of adjuvant arthritis. When examined at the peak of the acute inflammatory response, 5 days after adjuvant treatment, zinc given daily (2 mg/kg, intraperitoneally) increased serum ceruloplasmin levels by 2.0 times that found in nonarthritic rats and 1.2 times that found in non-zinc-treated arthritic rats. 13-cis-Retinoic acid (160 mg/kg, orally) given daily increased serum ceruloplasmin 2.2 and 2.7 times that found in nontreated arthritic rats when given alone and with zinc (2 mg/kg, intraperitoneally), respectively. Reduction in the inflammatory response was measured by weight of the adjuvant-injected paw, 5 days after adjuvant was administered. The reduction in inflammation was 13 and 19-20% for 13-cis-retinoic acid and zinc, respectively, when given alone, and between 26 and 31% when the treatments were combined. Zinc markedly increased liver metallothionein levels whereas 13-cis-retinoic acid was a much less potent inducer of the protein in liver. The results are discussed in light of the probable physiological roles of both ceruloplasmin and metallothionein.

Changes in rat liver metallothionein and metallothionein mRNA induced by isopropanol.

Administration of isopropanol (1 ml/kg body weight) via the ip route significantly depressed the serum zinc concentration within 8 hr. A maximal increase in hepatic metallothionein was observed 16 hr after isopropanol. By 48 hr after treatment metallothionein levels in liver had returned to basal levels. The extent of metallothionein induction was comparable with that observed after ip administration of zinc. Plasma glucagon concentrations were significantly elevated 4 hr after isopropanol treatment. Adrenalectomy did not prevent the isopropanol-induced changes in serum zinc or hepatic metallothionein. This suggests a nonadrenal mechanism is responsible for the observed changes. To evaluate changes in metallothionein mRNA levels in liver, in vitro translation with the wheat germ system was used to evaluate translational activity. Analysis of the labeled metallothionein produced in vitro employed both covalent chromatography as well as SDS-polyacrylamide gel electrophoresis of carboxymethylated translation products. These methods suggested the maximum metallothionein mRNA level in total RNA extract occurred about 8 hr after administration of isopropanol. Similarly, when metallothionein mRNA levels were quantitated using dot blot hybridization to [32P]cDNA for mouse metallothionein I, maximum metallothionein mRNA appeared 8 hr after isopropanol administration. The overall response of these parameters in rats suggest that isopropanol administration leads to an inflammatory-like response that, with respect to zinc metabolism, has elements which are independent of the adrenal gland, but involve transcriptional regulation of the metallothionein gene in liver.

Toxicity of a vitamin D steroid to laying hens.

Two experiments were conducted with 56-week-old or 104-week-old Leghorn hens to determine if feeding vitamin D steroids in excess of requirement levels caused any marked affects on eggshell quality. In the first experiment caged hens had reduced feed consumption, egg shell quality, and egg production as early as 6 weeks after initially consuming a basal diet supplemented with 6.8 micrograms 1 alpha-hydroxycholecalciferol (1 alpha-OH-D3)/kg. The second experiment confirmed the previous results and showed that extensive weight loss occurred with continued feeding of 10 or 15 micrograms 1 alpha-OH-D3/kg diet. No adverse affects were observed in either experiment when the level of 1 alpha-OH-D3 supplementation was 5.0 micrograms/kg diet or less. No toxic effects were observed when the hormone precursor 25-OH-D3 was supplemented to diets at 6 or 12 micrograms/kg. It is suggested that the pathological effects observed are related to the potent calcium homeostatic properties of 1 alpha-OH-D3 that at elevated levels may cause aberrations in circulating calcium.

Endotoxin-induced changes in copper and zinc metabolism in the Syrian hamster.

The temporal response of zinc and copper metabolism to endotoxin administration was examined in Syrian hamsters over a 144-hour period. Serum copper was significantly elevated at 12, 24 and 72 hours after endotoxin, whereas serum zinc was reduced 4-48 hours after treatment. A brief elevation (8 hours) in liver copper concentration and a sustained (72 hours) increase in liver zinc concentration were also observed. The amount of zinc associated with liver metallothionein (MT) progressively increased with time, to a plateau by 24 hours and persisted at the elevated level until 72 hours after endotoxin treatment. In vitro translation of poly (A)+ RNA from liver polyribosomes showed that following endotoxin treatment MTmRNA activity was maximally elevated 6 hours after endotoxin administration and remained elevated 24 and 48 hours thereafter. Slab gel electrophoresis of serum proteins indicated changes in a stainable protein comigrating with purified ceruloplasmin after endotoxin administration. Pooled gingival tissue from endotoxin-treated hamsters demonstrated a consistently elevated copper content 12-144 hours after treatment. Endotoxin isolated from Bacteroides melaninogenicus was more effective in elevating gingival and serum copper and gingival zinc than Escherichia coli endotoxin. It was concluded that endotoxin administration elicits responses that result in enhanced metaollthionein mRNA activity. In addition, Cu and Zn concentrations in serum, liver and gingival tissue are influenced by different endotoxins to different degrees.

Induction of kidney metallothionein and metallothionein messenger RNA by zinc and cadmium.

The influence of a 7.7-mumole (0.5-mg) dose of parenteral zinc on the synthesis of metallothionein in rat kidney was examined. The amount of zinc bound to metallothionein was maximal 9--15 hours after zinc administration. Pulse labeling with [35S]cystine showed the rate of renal metallothionein was stimulated to a maximum 6 hours after zinc administration and declined thereafter. Total RNA was extracted by the guanidine thiocyanate procedure from kidneys that had been flash frozen in liquid N2. Polyadenylated RNA (mRNA) was isolated by oligo (dT)-cellulose chromatography. The mRNA was translated in a wheat germ system and newly synthesized metallothionein was isolated by activated thiol--Sepharose 4B chromatography. Metallothionein mRNA activity nearly doubled after zinc administration adn was closely correlated with the enhancement in synthetic rate of this protein in kidney. Actinomycin D administered prior to either zinc or cadmium completely blocked both the stimulation of metallothionein synthesis and mRNA activity found in kidney in response to these metals. The results suggest renal metallothionein is induced by zinc or cadmium through a mechanism that requires altered expression of the metallothionein gene(s).