Sistemas de aleitamento artificial para bezerros em condições tropicais / Artificial feeding systems for calves in tropical conditions

Objetivou-se avaliar o desempenho bioeconômico de bezerros, nos primeiros 60 dias de vida, submetidos a três sistemas de aleitamento. Foram utilizados 24 bezerros (Holandês x Guzerá), sendo 12 machos e 12 fêmeas, com peso inicial de 32,25±4,8kg para as fêmeas e 36,92±6,8kg para os machos. Os animais foram distribuídos em delineamento inteiramente ao acaso, em esquema fatorial (3 x 2). Os bezerros receberam água à vontade e seis litros de sucedâneo lácteo por dia, durante 60 dias, em três estratégias diferentes, denominadas sistema de aleitamento (SA30: 3 litros de sucedâneo lácteo, duas vezes ao dia, até 30 dias de idade; SA45: 3 litros de sucedâneo lácteo, duas vezes ao dia, até 45 dias de idade; SA49: 3 litros de sucedâneo lácteo, duas vezes ao dia, até 49 dias de idade). Os sistemas de aleitamento estudados não apresentaram diferença estatística (P>0,05) para o consumo e a digestibilidade de nutrientes, com exceção para o consumo de matéria orgânica (MO) e extrato etéreo (EE). Verificou-se interação (P<0,05) entre o sistema de aleitamento e a classe sexual para os consumos de MO e EE, bem como para o ganho médio diário, em que os machos do SA 49 apresentaram maiores médias em relação ao SA 30. O desempenho bioeconômico de bezerros machos do sistema de aleitamento 49 foi superior e apresentou a melhor relação custo-benefício entre os sistemas estudados.(AU)

The objective of this study was to evaluate the bioeconomic performance of calves in the first 60 days of life submitted to three feeding systems. Twenty-four calves (Dutch x Guzerá) were used, 12 males and 12 females, with initial weight of 32.25±4.8kg for females and 36.92±6.8kg for males. The animals were distributed in a completely randomized design, in a factorial scheme (3 x 2). The calves received water at will and six liters of milk replacer a day for 60 days in three different strategies, called the suckling system (SA-30: 3 liters of milk replacer, twice a day until 30 days of age; SA-45: 3 liters of milk replacer, twice a day until 45 days of age; SA-49: 3 liters of milk replacer, twice daily up to 49 days old). The lactation systems studied did not present statistical difference (P>0.05) for the consumption and digestibility of nutrients, except for organic matter (OM) and ethereal extract (EE). There was an interaction (P<0.05) between the suckling system and sexual class for the OM and EE intakes, as well as for the average daily gain, in which HS 49 males presented higher averages in relation to SA 30. The bioeconomic performance of male calves from the lactation system 49 was superior and presented the best cost-benefit ratio among the systems studied.(AU)

Viabilidade econômica do uso de fontes lipídicas na dieta de vacas em lactação / Economic viability of using lipidic sources in the diet of lactating cows

Objetivou-se, com este estudo, avaliar a viabilidade econômica do uso de diferentes fontes lipídicas na dieta. O trabalho foi implantado em uma área de 42ha, dividida em oito piquetes, formada de Brachiaria brizantha. Utilizaram-se 12 vacas mestiças Holandês x Zebu, distribuídas em três quadrados latinos 4 x 4. Os quatro tratamentos foram constituídos de diferentes fontes lipídicas, como se segue: dieta sem fonte extra de lipídeos; dieta com caroço de algodão; dieta com óleo de soja; e dieta com óleo de soja de fritura. As dietas foram calculadas para suprir as exigências de mantença e produção de 15kg de leite/dia, com 3,5% de gordura. Utilizaram-se, para efeito de estudo da análise econômica, dois indicadores econômicos, valor presente líquido e taxa interna de retorno. O custo total por animal e por litro de leite produzido aumentou com a utilização das fontes lipídicas na dieta. Todos os tratamentos apresentaram valores positivos para lucro por animal, sendo observados valores de R$ 2,82; R$ 2,68; R$ 2,39 e R$ 2,09 para os tratamentos ausente de fonte extra de lipídeos; óleo de soja de fritura; caroço de algodão e óleo de soja, respectivamente. A taxa interna de retorno foi mais vantajosa quando não se utilizou fonte lipídica na dieta, o que demonstra que este tratamento é economicamente mais interessante para um investidor, gerando 0,73% ao mês. O cálculo do valor presente líquido demonstra que este investimento é viável para todos os tratamentos com taxa de 6% de desconto. O tratamento sem fonte extra de lipídeos apresentou menor custo de produção e, consequentemente, melhor relação custo-benefício. Para taxas de retorno do valor presente líquido de 10 e 12%, a produção leiteira torna-se inviável para todos os tratamentos.

The objective was to evaluate the economic viability of using different lipid sources in the diet of lactating cows. The experiment was done in an area of 42ha, divided into eight paddocks with an average size of approximately 5.3ha each, consisting of Brachiaria brizantha. Twelve Holstein x Zebu crossbred cows were distributed in three 4 x 4 Latin Squares. The four treatments consisted of different lipid sources, as follows: no extra source of dietary lipids, diet with cottonseed, diet with soybean oil, and diet with soybean frying oil. The diets were formulated to meet the requirements for maintenance and production of 15 kg/day, with 3.5% fat. To study the effect of economic analysis, two economic indicators were used: net present value and internal return rate. A one year simulation was produced to study economic characteristics, computing the depreciation of facilities and machinery in this period. The total cost per animal and per liter of milk produced increased with the use of lipid sources in the diet. All treatments had positive values for profit per animal, with observed values of R$ 2.82, R$ 2.68, R$ 2.39 and R$ 2.09, for treatments without lipid source; soybean frying oil as lipid source; cottonseed as lipid source and soybean oil as lipid source, respectively. The internal return rate was more advantageous when no fat source was used in the diet, demonstrating that this treatment is economically better for an investor, generating 0.73% per month. The net present value calculation shows that this investment is viable for all treatments with a 6% discount rate. Treatment without lipid source had a lower production cost and therefore is more cost-effective. Milk production is not viable for any treatment with return rates of the net present value of 10 and 12%.

Comportamento ingestivo de vacas em lactação alimentadas com cana-de-açúcar ou feno da parte aérea da mandioca / Ingestive behavior of dairy cows fed sugar cane or cassava aerial portion hay

Avaliou-se o efeito de dietas com cana-de-açúcar ou diferentes porcentagens de inclusão de feno da parte aérea da mandioca (FPAM) sobre o comportamento ingestivo de vacas leiteiras. Utilizaram-se 16 vacas, sendo 12 em lactação e quatro secas, fistuladas, distribuídas em quatro quadrados latinos 4x4. As dietas foram formuladas na tentativa de serem isoenergéticas, com quatro porcentagens de FPAM na dieta 0, 33, 67 e 100% da MS total da dieta, em substituição à cana-de-açúcar tratada com 1% de uma mistura de ureia e sulfato de amônio (9:1 partes). O comportamento ingestivo foi avaliado durante 24 horas consecutivas, sendo as observações efetuadas em intervalos de cinco minutos. Foi observado efeito quadrático (P<0,05) sobre os consumos de matéria seca e fibra em detergente neutro, por dia e por período de alimentação, sobre as atividades de alimentação, ruminação, mastigação e ócio, bem como sobre as eficiências de alimentação e ruminação. Não foi apresentada diferença (P>0,05) para os números e períodos de alimentação e ruminação. A avaliação do comportamento ingestivo constitui ferramenta de mensuração da quantidade e qualidade das dietas consumidas, uma vez que demonstra a resposta ingestiva dos bovinos à dieta fornecida.

The effect of diets containing sugar cane or different percentages of cassava aerial portion hay (FPAM) inclusion on dairy cows ingestion behavior was evaluated. Sixteen cows were used, including twelve lactating and four dry, which were fistulated and divided into four 4 x 4 Latin squares. Diets were formulated in an attempt to be isoenergetic, with four percentages of FPAM in the diet (0, 33, 67 and 100% of diet total DM), replacing the sugar cane treated with 1% of a mixture of urea and ammonium sulfate (9:1 parts). The ingestion behavior was evaluated for 24 consecutive hours, with observations at five minute intervals. A quadratic effect (P<0.05) on dry matter and neutral detergent fiber intake, per day and feeding period, on feeding, ruminating, chewing and idle activities, as well as on feeding and ruminating efficiencies, with no difference (P>0,05) for numbers and periods of feeding and rumination was observed. The ingestion behavior evaluation constitutes a tool for measuring the amount and quality of diets consumed, since it demonstrates their ingestion response by cattle diet provided.

Differential expression of alpha-2,3-sialyltransferases and alpha-1,3/4-fucosyltransferases regulates the levels of sialyl Lewis a and sialyl Lewis x in gastrointestinal carcinoma cells.

Sialyl Lewis x and sialyl Lewis a expression depends on sialyltransferases and fucosyltransferases. In this study, we screened for major variations of sialyltransferases and fucosyltransferases involved in the synthesis and regulation of sialyl Lewis x and sialyl Lewis a epitopes in gastrointestinal carcinoma cells. Our results show that expression of ST3Gal IV in several gastrointestinal cell lines is correlated with the expression of sialyl Lewis x at the cell surface. ST3Gal IV overexpressed in the gastric MKN45 cell line, showed exclusive enzymatic activity towards glycoproteins containing terminal Galbeta1-4GlcNAc structure. On the other hand, when ST3Gal III was overexpressed in MKN45, an increase in the expression levels of both sialyl Lewis epitopes was observed. ST3Gal III and ST3Gal IV lead to de novo synthesis of sialyl Lewis x determinant on different molecular weight glycoproteins of MKN45 cells suggesting that each enzyme used different substrates within the available glycoproteome. The final glycosylation step in sialyl Lewis x and sialyl Lewis a biosynthesis in MKN45 cell line was shown to be associated to FUT5, which efficiently fucosylated sialyl Lewis precursors on glycoproteins. Moreover we demonstrate that the expression of sialyl Lewis epitopes in the MKN45 was induced by cell confluence, which can be regarded as a model to study altered glycosylation during tumour progression. This increase was observed together with an increase in mRNA levels of ST3GAL3, FUT5 and FUT6, and a decrease in FUT4 transcript levels in MKN45 confluent cells, suggesting a possible control at the transcriptional level.

LexA-DNA bond strength by single molecule force spectroscopy.

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.

Structural studies of oligonucleotides containing G-quadruplex motifs using AFM.

G-quadruplex DNAs are cyclic arrays of four guanine bases binding by Hoogsteen hydrogen bonds, found in the telomeric regions of chromosomes and in transcriptional regulatory regions of several important oncogenes. Here, we used high resolution atomic force microscopy (AFM) to observe a specific guanine (G) tetrad mediated complex formation of oligonucleotides containing a G-quadruplex motifs (G-ODN) combined with a palindromic sequence under physiological extracellular conditions. These oligonucleotides have been investigated in correlation to their immunostimulatory effects. We observed structural dependence on ion concentration and G-ODN concentration, where high concentration self-assembled DNA networks were formed.

CDX2 is mutated in a colorectal cancer with normal APC/beta-catenin signaling.

The majority of human colorectal cancers have elevated beta-catenin/TCF regulated transcription due to either inactivating mutations of the APC tumor suppressor gene or activating mutations of beta-catenin. Surprisingly, one commonly used colorectal cancer cell line was found to have intact APC and beta-catenin and no demonstrable beta-catenin/TCF regulated transcription. However, this line did possess a truncating mutation in one allele of CDX2, a gene whose inactivation has recently been shown to cause colon tumorigenesis in mice. Expression of CDX2 was found to be induced by restoring expression of wild type APC in a colorectal cancer cell line. These findings raise the intriguing possibility that CDX2 contributes to APC's tumor suppressive effects.

Characterization of MAD2B and other mitotic spindle checkpoint genes.

Aneuploidy is a characteristic of the majority of human cancers, and recent work has suggested that mitotic checkpoint defects play a role in its development. To further explore this issue, we isolated a novel human gene, MAD2B (MAD2L2), which is homologous to the spindle checkpoint gene MAD2 (MAD2L1). We determined the chromosomal localization of it and other spindle checkpoint genes, including MAD1L1, MAD2, BUB3, TTK (MPS1L1), and CDC20. In addition, we resolved the genomic intron-exon structure of the human BUB1 gene. We then searched for mutations in these genes in a panel of 19 aneuploid colorectal tumors. No new mutations were identified, suggesting that genes yet to be discovered are responsible for most of the checkpoint defects observed in aneuploid cancers.

Identification of c-MYC as a target of the APC pathway.

The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.

A simplified system for generating recombinant adenoviruses.

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.

Homeosis and polyposis: a tale from the mouse.

Homeobox genes play essential roles in specifying the fates of different cell types during embryogenesis. In Drosophila, the homeotic gene caudal is important for the generation of posterior structures. In the mouse, the caudal homologue Cdx2 has been implicated in directing early processes in intestinal morphogenesis and in the maintenance of the differentiated phenotype. A recent study showed that Cdx2 null mutation was embryonically lethal, whereas Cdx2+/- mice developed multiple intestinal polyps in the proximal colon in addition to developmental defects. There are striking phenotypic similarities and differences between Cdx2+/- and other mice predisposed to intestinal neoplasia. The possible role of Cdx2 in human colorectal tumorigenesis is discussed.

Converting cancer genes into killer genes.

Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known.

Homozygous deletion map at 18q21.1 in pancreatic cancer.

Absolute genetic differences between neoplastic and nonneoplastic cells can be discerned at sites of homozygous deletions. These deletions are of critical interest because they might be useful in the identification of defective biochemical pathways in neoplastic cells, and subsequently for the development of new treatment strategies in human cancer. We identified an area at 18q21.1 involved by homozygous deletions in 30% of pancreatic carcinomas. To characterize the homozygous deletions, we constructed a detailed physical map of nearly 2 Mb, containing yeast artificial chromosomes, P1-derived artificial chromosomes, cosmids and 24 sequence-tagged sites. The homozygously deleted are contained a new candidate tumor-suppressor gene (DPC4). To date, 23 (64%) of 35 pancreatic carcinomas carry at least one homozygous deletion at a published locus. The study of the total gene content of these loci, facilitated by the sequence-tagged site markers and maps of these regions, should help to reveal the absolute biochemical differences between neoplastic and nonneoplastic cells for a common human tumor.

DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1.

About 90 percent of human pancreatic carcinomas show allelic loss at chromosome 18q. To identify candidate tumor suppressor genes on 18q, a panel of pancreatic carcinomas were analyzed for convergent sites of homozygous deletion. Twenty-five of 84 tumors had homozygous deletions at 18q21.1, a site that excludes DCC (a candidate suppressor gene for colorectal cancer) and includes DPC4, a gene similar in sequence to a Drosophila melanogaster gene (Mad) implicated in a transforming growth factor-beta (TGF-beta)-like signaling pathway. Potentially inactivating mutations in DPC4 were identified in six of 27 pancreatic carcinomas that did not have homozygous deletions at 18q21.1. These results identify DPC4 as a candidate tumor suppressor gene whose inactivation may play a role in pancreatic and possibly other human cancers.

Isolation of YAC insert sequences by representational difference analysis.

We present a method for the isolation of YAC insert sequences by representational difference analysis (RDA). To achieve maximal representation of the sequences, the amplicons were generated from a Mbol digestion product. RDA was performed using a 970 kb insert YAC clone. After two rounds of re-association and selective amplification 92% of the difference product represented sequences derived from the YAC insert. Twenty insert-specific sequence-tagged sites were readily defined. The difference product was also successfully used to isolate microsatellite markers, to identify clones from a human PAC library and as a chromosome painting probe in fluorescence in situ hybridization.

An integrated high-resolution physical map of the DPC/BRCA2 region at chromosome 13q12.

We identified a homozygous deletion in a pancreatic carcinoma (DPC) that localized to a 1-cM region at chromosome 13q12.3, which lay within the 6-cM locus of familial breast cancer susceptibility (BRCA-2). Here we present a physical map of the region, consisting of YAC, PAC, and cosmid contigs. The YAC contig comprises 16 clones that together span the entire BRCA2 region. The PAC contig comprises 22 clones that together span the DPC region. Seventy cosmid clones were localized within and near the DPC region. Thirty-five sequence-tagged sites were defined and localized within the map. The map indicates the size of the DPC region to be near 250 kb, and provides mapped and cloned resources for the search for the putative tumor suppressor gene(s) in the region.

Allelotype of pancreatic adenocarcinoma using xenograft enrichment.

p53 and MTS1 are known to be mutationally inactivated in pancreatic adenocarcinoma. Other tumor suppressor genes are likely also to play a role. To define chromosomal arms which may harbor additional tumor suppressor genes, we performed an extensive allelotype on pancreatic cancer utilizing a xenograft enrichment technique. Eighty-eight percent (28/32) of primary tumors gave rise to xenografts. Eighteen cases were used in a PCR-based allelotype using 283 polymorphic markers, over 2800 informative assays, and an average coverage of 4.1 informative markers per chromosomal arm per case. Highly frequent allelic loss (> 60%) was seen at chromosomes 1p, 9p, 17p, and 18q. Moderately frequent allelic loss (40-60%) was seen at 3p, 6p, 6q, 8p, 10q, 12q, 13q, 18p, 21q, and 22q. The average fractional allelic loss was 0.36. Allelic and sequence stability was demonstrated among 64 parallel and second-passage xenografts derived from 12 cases of pancreatic adenocarcinoma with the ascertainment of over 3000 single alleles. The findings were confirmed in primary tumors. In only two instances were discrepancies revealed between the allelic loss data obtained from corresponding parallel xenografts, probably due to the xenografting of minor subpopulations, reflecting genetic heterogeneity of the primary tumor.

Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals.

The broad spectrum of biological responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is believed to be due to the alteration in expression of TCDD-inducible genes. The aim of this study was to investigate the effects of TCDD on the in vivo tissue-specific expression of the recently identified TCDD-inducible cytochrome P450 CYP1B1 [Sutter et al. (1994) J. Biol. Chem., 269, 13092-13099] in Sprague-Dawley rats. We cloned the 5.0 kb rat homolog of CYP1B1 from a TCDD-treated rat liver cDNA library and showed that the rat and human CYP1B1 predicted amino acid sequences are 80% identical. RNA hybridization analysis showed that CYP1B1 is constitutively expressed in the adrenal glands and also in the testes of untreated rats. This tissue distribution suggests that CYP1B1 may be a physiological steroid hydroxylase. Seventy-two hours post-administration of 25 micrograms/kg body wt TCDD by gavage, steady-state levels of the 5.1 kb CYP1B1 RNA were increased > 50-fold in liver, and to a lesser extent in kidneys, lung, heart and ovaries. Average CYP1B1 RNA levels were significantly higher in the kidneys and livers of TCDD-treated females than in those from similarly treated males. In contrast, no significant sex-difference was observed in the levels of CYP1A1 in these tissues in TCDD-treated animals. In Sprague-Dawley rats, TCDD is a more potent hepatocarcinogen in females than in males. The induction of CYP1B1 in TCDD rat liver may be a contributing factor to the carcinogenic action of this persistent environmental pollutant.

Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region.

Homozygous deletions have been central to the discovery of several tumor-suppressor genes, but their finding has often been either serendipitous or the result of a directed search. A recently described technique [Lisitsyn, N., Lisitsyn, N. & Wigler, M. (1993) Science 259, 946-951] held out the potential to efficiently discover such events in an unbiased manner. Here we present the application of the representational difference analysis (RDA) to the study of cancer. We cloned two DNA fragments that identified a homozygous deletion in a human pancreatic adenocarcinoma, mapping to a 1-centimorgan region at chromosome 13q12.3 flanked by the markers D13S171 and D13S260. Interestingly, this lies within the 6-centimorgan region recently identified as the BRCA2 locus of heritable breast cancer susceptibility. This suggests that the same gene may be involved in multiple tumor types and that its function is that of a tumor suppressor rather than that of a dominant oncogene.