Feed efficiency metrics in growing pigs.

The objective of the present study was to quantify the interrelationships between different feed efficiency measures in growing pigs and characterize pigs divergent for a selection of these measures. The data set included data from 311 growing pigs between 42 and 91 d of age from 3 separate batches. Growth-related metrics available included midtest metabolic BW (BW), energy intake (EI), and ADG. Ratio efficiency traits included energy conversion ratio (ECR), Kleiber ratio (ADG/BW), relative growth rate (RGR), residual EI (REI), and residual daily gain (RDG). Residual intake and gain (RIG; i.e., a dual index of both REI and RDG) and residual midtest metabolic weight (RMW) were also calculated. Simple Pearson correlations were estimated between the growth and feed efficiency metrics. In litters with at least 3 pigs of each sex, pigs were separately stratified on each residual trait as high, medium, and low rank. Considerable interanimal variability existed in all metrics evaluated. Male pigs were superior to females for all metrics ( < 0.001) except for both BW and EI, where no sex differences were evident. Feed efficiency metrics improved as birth BW increased ( < 0.05) except for RGR, where the contrary was observed. Correlations between most growth and feed efficiency metrics were strong to moderate ( < 0.05). Low-REI pigs (i.e., more efficient) had lower EI and ECR and were superior for RIG ( < 0.001) compared with high- and medium-REI pigs. High-RDG pigs (i.e., more efficient) had greater BW gain and better ECR ( < 0.001) compared with medium- and low-RDG pigs. Residual EI and RIG were both superior ( < 0.001) in high-RDG pigs compared with medium- and low-RDG pigs. Energy conversion ratio, REI, and RIG were superior ( < 0.05) in high-RMW pigs (i.e., more efficient) compared with medium-RMW pigs. High-RIG pigs (i.e., more efficient) had lower EI ( < 0.01) and superior ECR for RDG and REI compared with medium- and low-RIG pigs. In general, most of the correlations among the feed efficiency traits investigated in this study were different from unity, indicating that each trait is depicting a different aspect of efficiency in pigs, although the moderate to strong correlations suggest that improvement in one trait would, on average, lead to improvements in the others. Pigs ranked as more efficient on residual traits such as REI consumed less energy for a similar BW gain, which would translate into an economic benefit for pig producers.

Erythropoietic protoporphyria a clinical and molecular study from Lebanon: Ferrochelatase a potential tumor suppressor gene in colon cancer.

Erythropoietic protoporphyria (EPP) is a rare cutaneous and systemic disease caused by mutations in the ferrochelatase gene (FECH). The molecular underpinnings of EPP in Middle Eastern populations and relative to other ethnic groups secondary to increased consanguinity are unknown. To understand the molecular pathogenesis of Middle Eastern EPP, we surveyed clinicopathological and molecular features in 6 large consanguineous families from Lebanon and Syria presenting with cutaneous and systemic features consistent with EPP. We observed 30% increased liver disease and 20% elevated end-stage liver complications in our EPP cohort compared to EPP patients previously reported elsewhere. In addition, Middle Eastern EPP patients in our cohort exhibited uniquely an increased incidence of colon cancer. Sequence analysis revealed 2 novel non-synonymous FECH mutations in the studied families designated p.M294T and p.I230M. In addition, FECH activity was significantly decreased (6%) in fibroblasts obtained from sun-exposed sites in a patient with p.M294T mutation, whereas in sharp contrast, protected sites from the same patient exhibited 54% activity for the gene. We also found that sun-exposed fibroblasts, relative to sun-protected and control fibroblasts, exhibited suppressed growth and atypical morphology in vitro, and that these effects were alleviated when the cells were co-cultured with sun-protected fibroblasts. Our findings on the increased incidence of colon cancer in EPP patients prompted us to survey FECH expression patterns in cancer. Using publicly available microarray datasets we found that FECH mRNA was largely significantly decreased in colon adenocarcinomas relative to normal colon tissues. Our findings suggest that families with autosomal recessive EPP should be screened more extensively for systemic involvement including liver diseases and colon cancer, and point to a previously unknown yet plausible tumor suppressor role for FECH in colon malignancy.

Modulator of heme biosynthesis induces apoptosis in leukemia cells.

OBJECTIVE: The purpose of this research is the investigation of the possible cause(s) of the dark-cell death phenomenon induced by 1,10-phenanthroline (Oph), a porphyrin biosynthesis modulator. SUMMARY BACKGROUND DATA: We have previously shown that porphyrin biosynthesis modulators, such as Oph, which is also an iron-chelating agent, enhance protoporphyrin IX (Proto) accumulation in mammalian neoplastic cells treated with delta-aminolevulinic acid (ALA). As a result of the enhanced Proto accumulation, a significant increase in photodynamic damage was observed under illumination. Also tetrapyrrole and heme-biosynthesis modulators have been shown to cause death in treated insect larvae in darkness, a phenomenon referred to as dark-cell death. Dark-cell death was also observed in Oph + ALA-treated transformed mammalian cells. METHODS: Neoplastic cells were treated with ALA, Oph, and ALA + Oph, and the following cell properties were investigated: growth arrest, membrane permeability, cell survival, nucleosomal cleavage, and cell cycle alterations. RESULTS: It was observed that Oph but not ALA induced growth arrest, in a T-cell leukemia line (MLA 144) as assessed by reduction in DNA synthesis. Exogenous Proto and isomers of Oph lacking the iron-chelating property of Oph also caused a dose-dependent inhibition of proliferation in MLA 144 cells. Although the plasma membrane of Oph-treated cells remained intact following 3 h of dark-incubation, the cells exhibited DNA internucleosomal cleavage, characteristic of cells undergoing apoptosis. Cell cycle analysis using the DNA intercalating dye propidium iodide (PI) coupled to flow cytometry, indicated that 81 +/- 5.6% of Oph-treated MLA 144 cells were apoptotic, with the majority of the cells arrested in the early S phase. On the other hand, treatment with either ALA or Proto did not alter the cell cycle. Also, using a double-labeling protocol with Hoechst 33342, and PI, and analysis by flow cytometry, Oph-treated cells were found to be 82% apoptotic after 3 h of dark-incubation. Apoptosis was reduced by 75% (p < 0.05) by the cytoplasmic protein synthesis inhibitor cycloheximide. CONCLUSIONS: These results indicate that in addition to enhancing Proto accumulation, the heme biosynthesis modulator Oph also induces growth arrest and apoptosis in transformed cells in darkness.

Regulation of myeloid growth and differentiation by the insulin-like growth factor I receptor.

Flow cytometry was used to examine the expression of type I insulin-like growth factor receptors (IGF-IR) on three types of human hematopoietic cells that represent different stages of myeloid lineage development. Both HL-60 (promyeloid) and U-937 (monocytic) cells express abundant IGF-IR protein (> 79% cells positive for the IGF-IR), whereas KG-1 myeloblasts express negligible levels of IGF-IR (< 1% IGF-IR-positive cells). Exogenous IGF-I, IGF-II, and an IGF-I analog that binds poorly to IGF-binding protein-3 (des-IGF-I) increased DNA synthesis of HL-60 and U-937 cells in a dose-dependent (1-25 ng/ml) fashion by 2- to 4-fold in serum-free medium, whereas KG-1 cells did not respond to any of these growth factors. The IGF-induced increase in proliferation of HL-60 promyeloid cells was inhibited by soluble IGF-binding protein-3 (500 ng/ml) when these cells were stimulated with 10 ng/ml of either IGF-I (53 +/- 8%) or IGF-II (59 +/- 8%), but not with des-IGF-I (3 +/- 1%). In contrast, the anti-IGF-IR monoclonal antibody (mAb; alpha IR-3) inhibited the DNA synthesis caused by 10 ng/ml exogenous IGF-I (67 +/- 6%), IGF-II (72 +/- 8%), and des-IGF-1 (82 +/- 9%). Proliferation of KG-1 myeloblasts, however, was neither stimulated by the IGFs nor inhibited by the anti-IGF-IR mAb. In the absence of exogenous IGF-I, the mAb directed against the IGF-IR significantly suppressed basal DNA synthesis of HL-60 promyeloid (72 +/- 5%) and U-937 monocytic (39 +/- 7%) cells, but did not affect DNA synthesis of KG-1 myeloblasts (8 +/- 1%) compared to an isotype-matched control mAb. Similarly, the alpha IR-3 mAb abrogated vitamin D3-induced differentiation of the HL-60 cells into macrophages in serum-free medium, as assessed by expression of the leucam surface protein, CD11b. As the alpha IR-3 mAb inhibits DNA synthesis in the presence and absence of exogenous IGF-I on receptor-bearing cells, but not IGF-IR-negative cells, these data demonstrate that both endocrine and autocrine IGF-I are potent growth factors in human myeloid cells where expression of the surface receptor, rather than the ligand, is the critical control element. More importantly, these data support the hypothesis that autocrine IGF-I may play a significant role in the differentiation of promyeloid cells into macrophages.

Enhancement of coproporphyrinogen III transport into isolated transformed leukocyte mitochondria by ATP.

It has been proposed that in normal animal cells, biosynthesis of the heme precursor protoporphyrin IX (Proto) requires cooperation between the mitochondria and cytoplasm [S. Granick (1967) in Biochemistry of the Chloroplast (Goodwin, T. W., Ed.), pp. 373-410, Academic Press, NY]. In this work, the conversion of ALA to Coprogen in the cytoplasm of MLA 144 cells (a retrovirally transformed gibbon ape leukemic T cell line) is demonstrated. This in turn indicates that the intracellular localization of Coprogen biosynthesis in transformed animal cells is similar to that proposed for normal animal cells. It is also shown that in MLA 144 cells, after ALA conversion to Coprogen in the cytoplasm, Coprogen is transported into the mitochondria via an ATP-dependent process. The possible involvement of the mitochondrial peripheral-type benzodiazepine receptor (M-PBR) in Coprogen transport into mitochondria is discussed.

Induction of tumor necrosis by delta-aminolevulinic acid and 1,10-phenanthroline photodynamic therapy.

delta-Aminolevulinic acid (ALA) causes cells to accumulate protoporphyrin IX (Proto) and heme. Exposure to light in vitro causes intracellular Proto to initiate formation of singlet oxygen molecules, leading to self-destruction. This photoactivated destruction by ALA in vitro is enhanced by addition of the tetrapyrrole modulator 1,10-phenanthroline (Oph), which increases cellular accumulation of Proto. Here we significantly extend this idea by evaluating the efficacy of ALA and Oph photodynamic therapy of solid tumors in vivo. Methylcholanthrene-induced fibrosarcoma (Meth-A) cells were used, which lead to the formation of solid tumors when implanted into syngeneic recipients. Initially, suspensions of Meth-A cells were treated in vitro with combinations of ALA and Oph. Meth-A cells in suspension accumulated 6-fold greater amounts of Proto (P < 0.05) after 3-h incubation with ALA and Oph than when incubated with ALA alone, and were also more susceptible to subsequent photoactivated cell lysis in vitro. Similarly, solid Meth-A tumors grown in syngeneic BALB/c mice accumulated significant (P < 0.05) amounts of Proto 3 h after in vivo treatment with ALA, and Oph synergized with ALA to significantly (P < 0.05) enhance the induction of Proto in these tumors. ALA and Oph-based phototreatment of mice bearing Meth-A solid tumors resulted in necrosis of tumors, as determined by a significant reduction in both size and histopathology, with little damage to surrounding normal tissue. These data directly demonstrate the experimental usefulness of Proto modulators for ALA-based photodynamic therapy in the treatment of solid tumors in vivo and provide a rationale for their potential application in a multitude of tumor types.

The colony-stimulating factors induce expression of insulin-like growth factor-I messenger ribonucleic acid during hematopoiesis.

Murine bone marrow cells cultured in the presence of colony-stimulating factor-1 (CSF-1) showed coordinate induction of insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) during the differentiation process, and these transcripts increased approximately 50- to 75-fold over the virtually negligible levels measured in freshly isolated bone marrow. In contrast, transcripts for the IGF-I receptor were evident in freshly isolated rat bone marrow cells and showed a 50% down-regulation during differentiation. Addition of a variety of single lineage and multilineage CSFs, including CSF-1, interleukin-3, granulocyte-macrophage-CSF, and granulocyte-CSF to mouse bone marrow cultures revealed that induction of IGF-I mRNA is a universal feature of differentiation with these CSFs, although IGF-I transcripts are at least 10- to 20-fold higher in CSF-1- and interleukin-3-differentiated lineages than in other cultures. The IGF-I induced by CSF-1 was biologically active because a natural ligand of IGF-I, IGF-binding protein-3, caused significant down-regulation of cellular proliferation, and this could be reversed by the addition of exogenous IGF-I. In addition, whereas IGF-I mRNA could be detected in resident peritoneal macrophages, these transcripts were increased 6-fold after a local injection of thioglycollate, a stimulus that induces macrophage proliferation and differentiation in vivo. These results show that CSFs induce expression of the growth factor IGF-I during differentiation of hematopoietic cells into multiple myeloid lineages and that this endogenously produced IGF-I is also a growth factor for hematopoietic cells. The induction of IGF-I mRNA during hematopoiesis should provide a new approach to understanding the expression of this gene during development and differentiation.

Interferon-gamma inhibits macrophage insulin-like growth factor-I synthesis at the transcriptional level.

Spontaneous production of insulin-like growth factor-I (IGF-I) by inflammatory macrophages contributes to aberrant wound healing, but little is known about regulation of IGF-I synthesis in myeloid cells. The T cell-derived cytokine interferon-gamma (IFN gamma) inhibits several fibrogenic and angiogenic components of the wound-healing response. We have used metabolic labeling of primary colony stimulating factor-1 (CSF-1)-derived macrophages and a transformed macrophage cell line (PU5-1R) followed by immunoprecipitation to demonstrate that synthesis of the 17 kilodalton (kDa) prepro-IGF-I protein by these cells is substantially inhibited by IFN gamma. An exon 4 IGF-I/beta-actin riboprobe expression cassette was used in RNase protection assays to show that IFN gamma also reduces steady state levels of IGF-I mRNA in three different populations of macrophages in a time- and dose-dependent manner. This effect is specific for IFN gamma because neither the IFNs-alpha/beta nor lipopolysaccharide (LPS) affects expression of steady state IGF-I transcripts. Down-regulation of IGF-I mRNA by IFN gamma is dependent on de novo protein synthesis and is abrogated by coculture with cycloheximide. Nuclear run-on assays revealed that elongation of IGF-I transcripts is absent in fresh bone marrow cells but is induced several-fold after cells are cultured for 6 days with CSF-1. Treatment of these CSF-1-derived macrophages with IFN gamma for 6 h substantially inhibits synthesis of IGF-I mRNA. Studies on the decay of IGF-I mRNA in PU5-1R macrophages treated with an RNA polymerase inhibitor confirmed that the decline in IGF-I steady state mRNA in IFN gamma-treated cultures arises from an inhibition of transcription rather than from a reduction in mRNA stability. Since a variety of inflammatory mediators can induce expression of IGF-I in macrophages, inhibition of macrophage IGF-I synthesis by IFN gamma provides a mechanism by which leukocytes regulate levels of this growth factor in their microenvironment.

Murine macrophages express abundant insulin-like growth factor-I class I Ea and Eb transcripts.

Hemopoietic cells have been reported to synthesize insulin-like growth factor-I (IGF-I) messenger RNA (mRNA), but the relative contribution of specific cell lineages that express these transcripts remains unknown. Reverse transcription and amplification of complementary DNA (cDNA) by the polymerase chain reaction were used to characterize full-length IGF-I mRNA transcripts in murine hemopoietic cells. The identity of transcripts encoding the entire prepropeptide was confirmed by restriction endonuclease digestion, Southern blotting, cloning, and Sanger sequencing. Abundance of IGF-I mRNA transcripts was assessed both by Northern blotting and sensitive ribonuclease protection assays followed by quantification with Phosphor-Imager analysis. Whereas IGF-I cDNA transcripts could be detected in a variety of leukocytes after polymerase chain reaction amplification, IGF-I mRNA was negligible or nondetectable in T and B cell lines and in those tissues containing a predominance of these cell types (e.g. spleen and thymus) by Northern blotting and ribonuclease protection assays. In contrast, elicited peritoneal macrophages, a macrophage cell line, microglia, and bone marrow macrophages differentiated in vitro expressed abundant IGF-I mRNA transcripts, whereas neither a premyeloid cell line nor freshly isolated bone marrow cells expressed significant transcripts. The 5'-identity of macrophage IGF-I transcripts was established using an exon 2-derived IGF-I cDNA probe. All protected transcripts were foreshortened, indicating transcript initiation exclusively within exon 1, characteristic of extra-hepatic IGF-I mRNA. However, at the 3'-end, both IGF-I Ea (lacking exon 5) and IGF-I Eb (containing exon 5) mRNA transcripts were evident, with the Eb product being detected at levels similar to those present in hepatic cellular RNA. A large molecular size (26 kilodaltons) prepro-IGF-I peptide was also detected in macrophage cell lysates by Western blotting. Collectively, our observations show that: 1) among hemopoietic cells, myeloid rather than lymphoid cells are the major source of IGF-I; 2) macrophage IGF-I mRNA consists of class I Ea and Eb transcripts; 3) these transcripts are translated into protein; and 4) expression of IGF-I is directly associated with differentiation of bone marrow macrophages.

Photodestruction of tumor cells by induction of endogenous accumulation of protoporphyrin IX: enhancement by 1,10-phenanthroline.

Rapidly proliferating transformed mammalian cells can be photodestroyed in vitro upon inducing the accumulation of endogenous protoporphyrin IX (Proto). Proto biosynthesis and accumulation were triggered by manipulation of the porphyrin-heme biosynthetic pathway. Proto accumulation in cultured cells was induced by treatment with 1.0 mM delta-aminolevulinic acid (ALA), a naturally occurring 5-carbon amino acid, for 3.5 h. In darkness, significant Proto accumulation became evident within 3.5 h of incubation. In the light, the accumulated tetrapyrroles triggered destruction of treated cells within the first 30 min of illumination, probably via the rapid oxidation of cellular constituents by singlet oxygen. Protoporphyrin IX accumulation and specific cell lysis increased significantly by inclusion of 0.75 mM 1,10-phenanthroline (Oph), a tetrapyrrole biosynthesis modulator. Slower growing untransformed cells did not accumulate significant amounts of Proto following ALA and Oph treatment unless stimulated to proliferate with the mitogenic lectin Concanavalin A.