Are dual-purpose and male layer chickens more resilient against a low-protein-low-soybean diet than slow-growing broilers?

1. Although fattening dual-purpose types or male layer hybrid chickens appears more ethical than the common practice of culling day-old male layer chicks, the lower feed efficiency of these birds raises concerns. Replacing feed ingredients that compete with food production by those of lower value for human nutrition would be beneficial.2. Lohmann Dual (LD), a modern dual-purpose type, Lohmann Brown (LB), a male layer hybrid, and Hubbard JA 957 (HU), a slow-growing broiler type, were fattened for nine weeks on two diets (control or -20% crude protein; n = 6 × 12 birds). Growth, carcass and meat quality were analysed.3. Growth performance of HU exceeded that of LD and especially of LB. The growth depression caused by the low-protein diet fed to LD (-7%) was only half of that found in HU (-13%). The LD fed the control diet had the same feed efficiency as the HU fed the low-protein diet. Even the LB had a lower performance and feed efficiency with the low-protein diet in growth. There was a gradient in carcass properties (weight, dressing percentage, breast meat yield, breast proportion and breast angle) from HU to LD to LB, with some additional adverse effects of the low-protein diet especially in HU. There were some breed differences in fatty acid profile in the intramuscular fat.4. In conclusion, the dual-purpose type used complied with regulations for Swiss organic poultry systems in terms of growth rate and was found to respond less when fed a low-protein diet than the slow-growing broiler type. The LB males were inferior in all growth and carcass quality traits. Future studies need to determine the exact protein and amino acid requirements of dual-purpose and layer hybrid chickens and the economic feasibility of the systems, especially for organic farming.

CyclinD-CDK4/6 complexes phosphorylate CDC25A and regulate its stability.

The phosphatase CDC25A is a key regulator of cell cycle progression by dephosphorylating and activating cyclin-CDK complexes. CDC25A is an unstable protein expressed from G1 until mitosis. CDC25A overexpression, which can be caused by stabilization of the protein, accelerates the G1/S and G2/M transitions, leading to genomic instability and promoting tumorigenesis. Thus, controlling CDC25A protein levels by regulating its stability is a critical mechanism for timing cell cycle progression and to maintain genomic integrity. Herein, we show that CDC25A is phosphorylated on Ser40 throughout the cell cycle and that this phosphorylation is established during the progression from G1 to S phase. We demonstrate that CyclinD-CDK4/CDK6 complexes mediate the phosphorylation of CDC25A on Ser40 during G1 and that these complexes directly phosphorylate this residue in vitro. Importantly, we also find that CyclinD1-CDK4 decreases CDC25A stability in a ßTrCP-dependent manner and that Ser40 and Ser88 phosphorylations contribute to this regulation. Thus our results identify cyclinD-CDK4/6 complexes as novel regulators of CDC25A stability during G1 phase, generating a negative feedback loop allowing control of the G1/S transition.

In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery.

RNA interference (RNAi)-mediated gene silencing approaches appear very promising for therapies based on the targeted inhibition of disease-relevant genes. The major hurdle to the therapeutic development of RNAi strategies remains, however, the efficient delivery of the RNAi-inducing molecules, the short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to the target tissue. With respect to cancer treatment the development of efficient delivery methods into solid tumors appears as a critical issue. However, very few studies have addressed this problem. In this study we have investigated the contribution of electrically mediated delivery of siRNA into murine tumors stably expressing an enhanced green fluorescent protein (EGFP) target reporter gene. The silencing of EGFP gene expression was quantified over time by fluorescence imaging in the living animal. Our study indicates that electric field can be used as an efficient method for siRNA delivery and associated gene silencing into cells of solid tumors in vivo.

Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery.

Owing to their capacity to induce strong, sequence-specific, gene silencing in cells, short interfering RNAs (siRNAs) represent new potential therapeutic tools. This development requires, however, new safe and efficient in vivo siRNA delivery methods. In the present technical report, we show that electrically mediated siRNA transfer can suppress transgene expression in adult mice muscles. Using electropulsation for siRNA delivery opens the way for a targeted gene silencing on a broad range of tissues. Clinical applications of electropulsation for delivery of other classes of molecules are under trials. We reported that gene silencing was efficiently obtained in vivo in an adult mammal (mouse) with chemically synthesized siRNA after its electrical delivery. The associated gene silencing was followed on the same animal and lasted at least 11 days. Gene silencing was obtained in muscles not only on young adult mice but also on much older animals. No tissue damages were detected under our electrical conditions. Therefore, this method should provide an efficient approach for a localized delivery of siRNAs in various tissues and organs.

Interconnections between E2-dependent regulation of cell cycle progression and apoptosis in MCF-7 tumors growing on nude mice.

Growth of human breast adenocarcinoma MCF-7 cells as a tumor on nude mice is dependent on estrogen. It has been shown that estrogen withdrawal (EW) induces a partial regression of the tumor via an inhibition of cell proliferation and an induction of apoptosis. We investigated in this in vivo model the underlying molecular mechanisms of the hormone-dependent regulation of cell cycle machinery and apoptosis. We found that, 2 days after EW, the tumor protein levels of p21 rose, whereas those of Rb proteins decreased in parallel with the decrease in the proportion of tumor cells in S phase and the increase of the tumor apoptotic index. Between 3 and 7 days after EW, apoptosis was inhibited and tumor proliferation returned to the control value. There was a concomitant decline in p21 and an elevation of Rb tumor protein content. Slight variations of cyclin D protein level were observed in MCF-7 tumors over the time course following EW treatment. Bcl-2 overexpression not only inhibited apoptosis induced by EW but also modulated hormone-dependent cell cycle regulation. First, the analysis of phosphorylation status of Rb protein and the measurement of the proportion of tumor cells in S phase indicated that Bcl-2 overexpression results in a decrease of DNA synthesis induced by estradiol. Furthermore, after EW, Bcl-2-induced inhibition of hormone-dependent apoptosis was associated with an inhibition of Rb protein downregulation, a sustained level of p21 protein, and a prolonged inhibition of cell cycle progression. These results suggest that, in human hormone-dependent breast cancers, cross-talk exists between the signaling pathways which lead to regulation of cell cycle progression and apoptosis.

Human 76p: A new member of the gamma-tubulin-associated protein family.

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.

Identification of proliferation-induced genes in Arabidopsis thaliana. Characterization of a new member of the highly evolutionarily conserved histone H2A.F/Z variant subfamily.

The changes in gene expression associated with the reinitiation of cell division and subsequent progression through the cell cycle in Arabidopsis thaliana cell-suspension cultures were investigated. Partial synchronization of cells was achieved by a technique combining phosphate starvation and a transient treatment with the DNA replication inhibitor aphidicolin. Six cDNAs corresponding to genes highly induced in proliferating cells and showing cell-cycle-regulated expression were obtained by the mRNA differential display technique. Full-length cDNA clones (cH2BAt and cH2AvAt) corresponding to two of the display products were subsequently isolated. The cH2BAt clone codes for a novel histone H2B protein, whereas the cH2AvAt cDNA corresponds to a gene encoding a new member of the highly conserved histone H2A.F/Z subfamily of chromosomal proteins. Further studies indicated that H2AvAt mRNA expression is tightly correlated with cell proliferation in cell-suspension cultures, and that closely related analogs of the encoded protein exist in Arabidopsis. The implications of the conservation of histone H2A.F/Z variants in plants are discussed.

Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence.

In an Arabidopsis thaliana T87-C3 cell-suspension culture, entry into the growth-arrest phase is rapidly followed by a loss of cell viability. Three cDNA clones, SRG1, SRG2, and SRG3, corresponding to genes with transcripts that accumulate during these late phases, were isolated by the mRNA differential display method. Amino acid sequence analysis shows that the putative SRG1 protein is a new member of the Fe(II)/ascorbate oxidase superfamily, and that SRG2 codes for a protein with significant homology to beta-glucosidases. Significantly, all three SRG genes are expressed in senescing organs of Arbidopsis plants. Two previously characterized genes, SAG2 and SAG4, induced during natural senescence in Arabidopsis, were also found to be expressed in cell-suspension cultures and have expression kinetics similar to those observed for the SRG1 gene. Taken together these finding suggest that certain molecular events are common to both plant senescence and growth arrest in arabidopsis cell suspensions. Both internucleosomal cleavage of nDNA and an apparent compaction of chromatin, two characteristic features of programmed cell death in animal cells, have been observed in Arabidopsis cell cultures at a stage corresponding to loss of cell viability.

A method for the elimination of false positives generated by the mRNA differential display technique.

The recently described mRNA differential display method provides an attractive tool for the isolation of genes showing regulated expression in a variety of systems. A key step in this technique consists of the isolation of PCR-synthesized radioactive cDNAs corresponding to differentially expressed mRNAs. Here, we show that the purified cDNAs remain contaminated with unrelated cDNA sequences that may lead to the artifactual isolation of false positives in the subsequent steps of the method. A powerful assay for the detection and elimination of this contaminating material, allowing the specific isolation of clones corresponding to the regulated genes identified by the differential display, is provided.

Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts.

A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro. These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.

Conserved epitopes on plant H1 histones recognized by monoclonal antibodies.

A series of monoclonal antibodies specific for distinct regions of H1 histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.

Strand-specific viral DNA synthesis in purified viroplasms isolated from turnip leaves infected with cauliflower mosaic virus.

There is some evidence that two steps are involved in the DNA replication of cauliflower mosaic virus (CaMV): the first one may occur in the nucleus and the second one in the cytoplasm of infected cells. The latter would correspond to the reverse transcription step recently proposed in the model of the viral life cycle, and could occur in the viroplasms which are CaMV-induced cytoplasmic inclusion bodies. In order to test whether viroplasms are capable of DNA synthesis and to characterize the associated enzymatic activities, we developed an extensive purification method for these organelles. Such isolated viroplasms are indeed able to incorporate radioactive precursors into exclusively viral-specific sequences without added template primer. Hybridization of sequences labeled in viroplasms to cloned CaMV DNA shows that the DNA synthesis occurs throughout the whole viral genome and has marked strand specificity; neosynthesized molecules are of minus polarity, i.e., complementary to the large viral transcript (35 S RNA). Moreover, during the purification of viroplasms, the poly(rC)-directed DNA synthesis activity, which is specific to infected plants, is preferentially retained.