Influence of feed form and source of soybean meal on growth performance, nutrient retention, and digestive organ size of broilers. 2. Battery study.

Two experiments were conducted to determine the apparent ileal digestibility (AID) of the amino acids (AA) of 4 commercial soybean meals (SBM) from the United States (USA-1, 48.1% CP and USA-2, 46.2% CP), Brazil (BRA, 47.6% CP), and Argentina (ARG, 46.3% CP) and the effects of the inclusion of these SBM in diets in mash, crumble, or pellet form on growth performance, total tract apparent retention of nutrients, and digestive organ size in broilers reared in cages from 1 to 25 d of age. In experiment 1, the AID of Lys was higher (P < 0.05) for the USA-2 than for the BRA SBM, with the SBM from USA-1 and ARG being intermediate. In experiment 2, 12 diets were arranged as a 3 × 4 factorial with 3 feed forms (mash, crumbles, and pellets) and the 4 sources of SBM used in experiment 1. The feeds were isonutritive and the AID of the AA of the SBM obtained in experiment 1 was used for diet formulation. Broilers fed mash had lower (P < 0.001) ADFI and ADG and poorer (P < 0.001) feed-to-gain ratio than broilers fed crumbles or pellets but source of SBM did not affect growth performance. Nitrogen retention was higher (P < 0.01) in birds fed mash than in birds fed crumbles or pellets at all ages. The total tract apparent retention of nutrients was lower (P < 0.05) for the BRA and ARG SBM diets than for the USA-1 and USA-2 SBM diets. Gizzard empty relative weight (% BW) was higher and gizzard pH lower for broilers fed mash than for broilers fed crumbles or pellets (P < 0.001). The results indicate that crumbling or pelleting of the diets improved growth performance of broilers from 1 to 25 d of age. Diets formulated with analyzed rather than calculated AID of AA of the SBM sources resulted in similar broiler performance.

Proteolytic digestion of bacterial inclusion body proteins during dynamic transition between soluble and insoluble forms.

Inclusion bodies formed by two closely related hybrid proteins, namely VP1LAC and LACVP1, have been compared during their building in Escherichia coli. Features of these proteins are determinant of aggregation rates and protein composition of the bodies, generating insoluble particles with distinguishable volume evolution. Interestingly, in LACVP1 and less perceptibly in VP1LAC bodies, an important fraction of the aggregated polypeptide is lost at a given stage of body construction. Stable degradation intermediates of the more fragile LACVP1 are concomitantly found embedded in the bodies. When recombinant protein synthesis is arrested in growing cells, the amount of aggregated protein drops while the amount of soluble protein undergoes a sudden rise before proteolysis. This indicates an architectural plasticity during the in vivo building of the studied inclusion bodies by a dynamic transition between soluble and insoluble forms of the recombinant proteins involved. During this transition, protease-sensitive polypeptides can suffer an efficient proteolytic attack and the resulting fragments further aggregate as inclusion body components.

Reversible activation of a cryptic cleavage site within E. coli beta-galactosidase in beta-galactosidase fusion proteins.

The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of beta-galactosidase and produced in E. coli from two related expression vectors. One of these vectors, carries a 429 bp DNA segment encoding the N-terminus peptide of VP60, and directs the synthesis of a larger fusion that contains the entire viral protein. Both fusion proteins are efficiently cleaved at a presumed trypsin-like target site within the carboxy moiety of beta-galactosidase (Arg 611-Thr 612), which is activated by the presence of the viral partner. In the larger fusion, VP60 is released by a cleavage within the linker region that affects about 10% of the chimeric proteins. In this situation, the resulting beta-galactosidase-like fragment recovers its natural proteolytic stability. These results prove that cryptic cleavage sites in beta-galactosidase can be efficiently activated in a fusion protein and suggest that this activation is based on reversible steric constraints generated by the fusion partner.

Pharmacological and biochemical interactions between opioids and cannabinoids.

Opioids and cannabinoids are among the most widely consumed drugs of abuse in humans. A number of studies have shown that both types of drugs share several pharmacological properties, including hypothermia, sedation, hypotension, inhibition of both intestinal motility and locomotor activity and, in particular, antinociception. Moreover, phenomena of cross-tolerance or mutual potentiation of some of these pharmacological effects have been reported. In recent years, these phenomena have supported the possible existence of functional links in the mechanisms of action of both types of drugs. The present review addresses the recent advances in the study of pharmacological interactions between opioids and cannabinoids, focusing on two aspects: antinociception and drug addiction. The potential biochemical mechanisms involved in these pharmacological interactions are also discussed together with possible therapeutic implications of opioid-cannabinoid interactions.

[Cystic ureteritis in a kidney transplantation candidate]. / Ureteritis quística en paciente candidata a trasplante renal.

Cystic ureteritis is a very uncommon pathology, whose pathogenesis is not well established. It is usually asociated with chronic infectious factors. It presents unspecific symptoms but characteristic radiologic findings. There is not an especific treatment for this disease. Kidney transplant is the final pathway for patients with chronic renal failure. We report a case of cystic ureteritis diagnosed during pre-transplant study.

Organization of the CYP1A cluster on human chromosome 15: implications for gene regulation.

The sequence and organization of the CYP1A cluster on human chromosome 15 was determined. A human genomic clone from a BAC library, containing both CYP1A1 and CYP1A2 genes, was isolated and sequenced. The results of Southern blot analysis using human genomic DNA were compatible with the structure of the BAC clone. The CYP1A1 and CYP1A2 genes are separated by a 23 kb segment that contains no other open reading frames. The CYP1A1 and CYP1A2 genes are in opposite orientation, revealing that the 5' flanking region is in common between the two genes. Analysis of the sequence obtained revealed the presence of xenobiotic response elements (XREs) previously reported for CYP1A1 and CYP1A2 and several additional consensus sequences for putative XREs. The presence of all the XREs upstream of both genes suggest that some of the regulatory elements known to control CYP1A1 gene expression, could also control CYP1A2 gene expression.

Extinction of cocaine self-administration produces a differential time-related regulation of proenkephalin gene expression in rat brain.

The purpose of this study was to examine the time course effects of extinction of cocaine self-administration behavior on proenkephalin (PENK) gene expression in caudate-putamen nucleus (ST), nucleus accumbens (Acc), olfactory tubercle (Tu), piriform cortex (Pir), ventromedial hypothalamic nucleus (VMN), and central amygdala (Ce) as measured by in situ hybridization histochemistry. Seventy-two littermate male Lewis rats were randomly assigned in triads to one of three conditions: (1) contingent intravenous self-administration of 1 mg/kg/injection of cocaine (CONT); (2) noncontingent injections of either 1 mg/kg/injection of cocaine (NONCONT); or (3) saline yoked (SALINE) to the intake of the self-administering subject. The self-administering rats were trained to self-administer cocaine under a FR5 schedule of reinforcement for a minimum of 3 weeks. After stable baseline levels of drug intake had been reached, saline was substituted for drug. Following this first extinction period, cocaine self-administration was reinstated for an additional period of 2 weeks. Immediately after cessation of the last session of cocaine self-administration (day 0) and 1-, 5-, and 10-day after the second extinction period, animal brains in each triad were removed to be processed for in situ hybridization. PENK mRNA levels were significantly higher in the cocaine groups when compared with SALINE group in the ST, Acc, Pir, and Tu regions on days 0, 1, 5, and 10 of the extinction and lower in the Ce region of CONT group when compared to NONCONT and SALINE groups on days 1, 5, and 10 of the extinction period. In the VMN nucleus, PENK mRNA content in CONT group versus NONCONT and SALINE groups was also lower, but there were statistically significant differences only on day 5. These results suggest that changes in PENK gene expression after contingent cocaine administration might be involved in cocaine withdrawal states.

Repeated administration of delta9-tetrahydrocannabinol produces a differential time related responsiveness on proenkephalin, proopiomelanocortin and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat.

The purpose of the present study was to explore the time related effects of repeated administration of delta9-tetrahydrocannabinol on opioid and corticotropin releasing factor gene expression in the hypothalamus and pituitary gland of the rat. By using in situ hybridization histochemistry, the effects of delta9-tetrahydrocannabinol (THC, 5 mg/kg per day; i.p.) were examined after 1, 3, 7 and 14 days of repeated administration on; (1) proenkephalin gene expression in the paraventricular (PVN) and ventromedial nuclei (VMN) of the hypothalamus, (2) proopiomelanocortin gene expression in the arcuate nucleus (ARC) of the hypothalamus and anterior (AL) and intermediate lobe (IL) of the pituitary gland, and (3) corticotropin releasing factor gene expression in the PVN. The results revealed that, in most of the hypothalamic and pituitary regions examined, repeated cannabinoid administration upregulates opioid and corticotropin releasing factor gene expression. However, the onset, the degree of magnitude of gene expression reached and the time related effects produced by repeated administration with delta9-tetrahydrocannabinol are dependent upon the brain and pituitary regions examined. Taken together, the results of the present study suggest that cannabinoids produce a time related differential responsiveness in opioid and corticotropin releasing factor gene expression, in areas of the hypothalamus and pituitary that may be related, at least in part, to a molecular integrative response to behavioral, endocrine and neurochemical alterations that occur in cannabinoid drug abuse.

Chronic administration of cannabinoids regulates proenkephalin mRNA levels in selected regions of the rat brain.

This study was designed to examine the interactions between the cannabinoid and enkephalinergic systems in the rat brain. To this aim, we have examined the effects of subchronic (5 days) administration (10 mg.kg-1.day-1; i.p.) of delta 9 -tetrahydrocannabinol (THC) or R-methanandamide (AM356) and chronic (18 days) administration with the synthetic cannabinoid receptor agonist CP-55,940 (1 mg.kg-1.day-1; i.p) on proenkephalin (PENK) mRNA levels in several brain regions of the rat. Twenty micrometer brain sections from striatum, nucleus accumbens, paraventricular nucleus, ventromedial nucleus, periaqueductal grey matter and mammillary nucleus were hybridized with an oligonucleotide probe complementary to PENK using in situ hybridization technique. Subchronic administration of THC or AM356 increased PENK mRNA levels in the ventromedial nucleus of the hypothalamus, (82%) and (39%), in the periaqueductal grey matter, (97%) and (49%), and mammillary nucleus, (43%) and (9%), respectively. In contrast, both drugs were without effect in the striatum and nucleus accumbens. On the other hand, chronic administration of CP-55,940 increased PENK mRNA levels in the striatum (44%), nucleus accumbens (25%), paraventricular (31%) and ventromedial nuclei of the hypothalamus (41%). These results revealed that chronic cannabinoid administration increases opioid gene expression in the rat central nervous system and suggest an interaction between the cannabinoid and enkephalinergic systems that may be part of a molecular integrative response to behavioral and neurochemical alterations that occur in cannabinoid drug abuse.

Time-dependent differences of repeated administration with Delta9-tetrahydrocannabinol in proenkephalin and cannabinoid receptor gene expression and G-protein activation by mu-opioid and CB1-cannabinoid receptors in the caudate-putamen.

The purpose of the present study was to examine the time-related effects of repeated administration of Delta9-tetrahydrocannabinol during 1, 3, 7 and 14 days on cannabinoid and mu-opioid receptor agonist-stimulated [35S]GTPgammaS binding, and CB1 cannabinoid receptor and proenkephalin gene expression in the caudate-putamen. Repeated administration with Delta9-tetrahydrocannabinol produced a time-related reduction in cannabinoid receptor synthesis and activation of signal transduction mechanisms in the caudate-putamen. Indeed, WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased 24% on day 1 and then progressively decreased finding a 42% decrease on day 14. Similarly, CB1 cannabinoid receptor mRNA levels decreased (22%) on day 3, reaching 50% reduction on day 7. In contrast, a pronounced increase is detected in DAMGO-stimulated [35S]GTPgammaS binding and proenkephalin mRNA levels in the caudate-putamen. The highest degree of increase was reached on day 7 of the treatment (35% of proenkephalin mRNA levels and 62% of DAMGO-stimulated [35S]GTPgammaS binding) and then values slightly decreased on day 14. Taken together, the results of the present study indicate that, in the caudate-putamen, repeated administration with Delta9-tetrahydrocannabinol produces a time-related increase in proenkephalin gene expression and mu-opioid receptor activation of G-proteins, and a time-related decrease in CB1 cannabinoid receptor gene expression and reduction in CB1 cannabinoid receptor activation of G-proteins. These results also suggest a possible interaction between the cannabinoid and opioid systems in the caudate-putamen which may be potentially relevant in the understanding of the alterations of motor behavior that occur after prolonged exposure to cannabinoids.

Antigenicity of a viral peptide displayed on beta-galactosidase fusion proteins is influenced by the presence of the homologous partner protein.

Several beta-galactosidase fusion proteins have been constructed containing the entire VP1 protein from foot-and-mouth disease virus (FMDV) [Corchero et al. (1996) J. Biotechnol. in press]. The antigenicity of the major immunodominant site A (13 amino acids in length) within the VP1 protein has been studied in competitive ELISA using a panel of seven monoclonal antibodies elicited against the whole virus and recognizing B-cell epitopes within this site. None of the fusion proteins is able to reproduce the antigenic profile of FMDV, all of them being less immunoreactive than the virus particles. On the other hand, significant differences in the reactivity of site A are displayed on the different fusion proteins, being for some antibodies about 10-fold. This indicates that the reactivity of a small peptide included in its natural place inside the heterologous domain can be significantly influenced by the position of the homologous partner in the fusion protein.

Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria.

Time-dependent aggregation of a plasmid-encoded beta-galactosidase fusion protein, VP1LAC, has been carefully monitored during its high-rate synthesis in Escherichia coli. Immediately after recombinant gene induction, the full-length form of the protein steadily accumulates into rapidly growing cytoplasmic inclusion bodies. Their volume increases during at least 5 h at a rate of 0.4 micron3 h-1, while the average density remains constant. Protein VP1LAC accounts for about 90% of the aggregated protein throughout the building process. Minor components, such as DnaK and GroEL chaperones, have been identified in variable, but low concentrations. The homogeneous distribution of inclusion bodies among the cell population and the coexistence of large, still growing bodies with newly appearing aggregates indicate that the aggregation cores are mutually exclusive, this fact being a main determinant of the in vivo dynamics of protein aggregation.

A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli.

The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype C1 virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.

Heat-inactivation of plasmid-encoded CI857 repressor induces gene expression from Ind- lambda prophage in recombinant Escherichia coli.

We have observed significant cell lysis upon temperature up-shift of recombinant Escherichia coli cultures harboring CI857-repressed lambda-based expression vectors. This event, that becomes evident about 30-40 min after the heat shock, takes place when using the lambda promoter system in Ind- lysogenic strains, but not in others commonly employed for recombinant gene expression. These results strongly suggest that the thermosensitive CI857 repressor, encoded by the expression vector, competes with CI Ind- molecules for binding to the prophage operator region, allowing for expression of lytic genes from the integrated Ind- viral genome upon temperature up-shift. Transcription of viral lytic genes does not include unspecific expression of a reporter sulA::lacZ gene fusion carried in the prophage genome. These results prompt, however, to carefully evaluate the limitations of expression systems based on pL/pR-CI857 in bacterial strains modified through lambda Ind- gene transfer vehicles.

Opioid and cannabinoid receptor-mediated regulation of the increase in adrenocorticotropin hormone and corticosterone plasma concentrations induced by central administration of delta(9)-tetrahydrocannabinol in rats.

The purpose of this study was to investigate the cannabinoid and opioid mediated regulation on the effects of central Delta(9)-tetrahydrocannabinol (Delta(9)-THC) administration on hypothalamus-pituitary-adrenal (HPA) axis activity in the male rat. Intracerebroventricular (i.c.v.) administration of delta(9)-THC (25, 50, 100 microg/rat) markedly increased plasma adrenocorticotropin hormone (ACTH) and corticosterone concentrations. Time course effect studies revealed that both hormones secretion peaked at 60 min after Delta(9)-THC i.c.v. administration (50 microg/rat), decreased gradually and returned to baseline levels by 480 min. The i.c.v. administration of the specific cannabinoid receptor antagonist SR-141716A (3 microg/rat) significantly attenuated the increase of both hormones secretion induced by Delta(9)-THC (50 microg/rat). Nevertheless, higher doses (12.5 and 50 microg/rat) of this compound increased both ACTH and corticosterone plasma concentrations. Subcutaneous (s.c.) administration with the opiate receptor antagonist naloxone (0.3 mg/kg) was without effect but significantly diminished the increase of both hormones secretion induced by Delta(9)-THC (50 microg/rat). Taken together, these results indicate that opiate and cannabinoid receptors are involved in the activation of the HPA axis induced by Delta(9)-THC. Furthermore, the increase of ACTH and corticosterone secretion after the administration of higher doses of SR-141716A than those required to block such activation, suggests that endogenous cannabinoids are tonically inhibiting the release of both hormones or that this agonist-like activity may be part of an uncharacterized action of this compound not mediated by cannabinoid receptors.

Differential basal proenkephalin gene expression in dorsal striatum and nucleus accumbens, and vulnerability to morphine self-administration in Fischer 344 and Lewis rats.

We have previously shown that the acquisition rate of intravenous morphine self-administration under a fixed ratio one (FR1) schedule of reinforcement was greater in Lewis (LEW) than Fischer 344 (F344) rats. The purpose of the present experiment was to examine the relative motivational properties of morphine (1 mg/kg) or food under progressive ratio (PR) schedules of reinforcement in LEW and F344 rats. In addition, by using in situ hybridization histochemistry we have measured in both strains of rats the basal level of proenkephalin (PENK) gene expression in dorsal striatum and nucleus accumbens (NAcc). The results show that LEW rats responded to significantly higher breaking points (BPs) than F344 rats for intravenous morphine self-administration. In contrast, no differences were found in BPs for food pellets. Basal PENK mRNA levels were significantly higher in the dorsal striatum and nucleus accumbens of F344 than in LEW rats. Taken together, these results reveal a strain difference in the reinforcing efficacy of morphine and in the basal PENK gene expression in brain regions involved in the reinforcing actions of opiates. These data also suggest that the strain differences in opiate self-administration behavior found in this and other studies may be related, at least in part, to differences in basal opioid activity between LEW and F344 rats.

delta 9-Tetrahydrocannabinol increases proopiomelanocortin gene expression in the arcuate nucleus of the rat hypothalamus.

delta 9-Tetrahydrocannabinol, the main psychoactive component of cannabis, produces a large spectrum of pharmacological effects, many of which have been linked to interaction with the opioid system. The aim of this study was to examine the effects of delta 9-tetrahydrocannabinol on proopiomelanocortin (POMC) gene expression in the arcuate nucleus of the hypothalamus and anterior lobe of the pituitary. We report, for the first time, that a 5-day treatment with delta 9-tetrahydrocannabinol (5 mg/kg per day, i.p.) increased (38%) POMC mRNA levels in the arcuate nucleus of the hypothalamus but was without effect in the anterior lobe of the pituitary. These data indicate that delta 9-tetrahydrocannabinol stimulates opioid gene expression and regulates distinctively POMC in the hypothalamus and the anterior lobe of the pituitary in the rat.

The position of the heterologous domain can influence the solubility and proteolysis of beta-galactosidase fusion proteins in E. coli.

The VP1 protein (23 kDa) of the foot-and-mouth disease virus has been produced in MC1061 and BL21 E. coli strains as beta-galactosidase fusion proteins, joined to either the amino and/or the carboxy termini of the bacterial enzyme. In BL21, devoid of La protease, all the recombinant fusion proteins are produced at higher yields than in MC1061, and occur mainly as inclusion bodies. The fusion of VP1 at the carboxy terminus yields a protease-sensitive protein whose degradation releases a stable, enzymatically active polypeptide indistinguishable from the native beta-galactosidase. On the contrary, when the same viral domain is fused to the amino terminus, the resulting chimeric protein is resistant to proteolysis even in the soluble form. These data demonstrate that the position of the heterologous domain in beta-galactosidase fusion proteins would not be irrelevant since it can dramatically influence properties of biotechnological interest such as solubility and proteolytic resistance.

Chronic treatment with CP-55,940 regulates corticotropin releasing factor and proopiomelanocortin gene expression in the hypothalamus and pituitary gland of the rat.

The purpose of the present study was to explore the molecular mechanisms by which the cannabinoid system may interact with the hypothalamic-pituitary adrenal axis and the proopiomelanocortin opioid system. To this aim and by using in situ hybridization histochemistry, the effects of chronic (18 days) administration with the synthetic cannabinoid receptor agonist [(-)-cis-3-[2-hydroxy-4-(1,1,-dimethylheptyl)-phenyl]-trans-4(-3-h ydroxypropyl)cyclohexanol)], CP-55,940 (1 mg/kg/day; i.p.) on corticotropin releasing factor and proopiomelanocortin gene expression were examined in the paraventricular and arcuate nuclei of the hypothalamus and anterior and intermediate lobes of the pituitary gland in the rat. Chronic administration with CP-55,940 increased corticotropin releasing factor mRNA levels (41%) in the paraventricular nucleus and proopiomelanocortin mRNA levels in the arcuate nucleus (25%) and anterior lobe of the pituitary (30%), but decreased (28%) of proopiomelanocortin transcript amounts in the intermediate lobe of the pituitary. These results revealed that chronic cannabinoid administration enhances corticotropin releasing factor and proopiomelanocortin gene expression in the hypothalamus and anterior pituitary, a process that may be considered as part of a molecular integrative response to the stress associated to cannabinoid drug abuse.

delta-9-Tetrahydrocannabinol increases prodynorphin and proenkephalin gene expression in the spinal cord of the rat.

Hypoalgesia induced by cannabinoid drugs has been found to implicate the opioid system. The effect of five days treatment with delta-9-tetrahydrocannabinol (THC) was examined on prodynorphin (PDYN) and proenkephalin (PENK) gene expression in the spinal cord of male rats. PDYN and PENK gene expression was estimated measuring by northern blot analysis mRNA levels in the whole spinal cord, containing perikarya of these neurons. The subchronic treatment with THC (5 mg/kg/day; 5 days; i.p.) produced an increase in PDYN (39%) and PENK (34%) gene expression when compared with the vehicle treated group. These results suggest that the effects of THC in the spinal cord involve an increase in opioid activity, and therefore sustain the hypothesis of an interaction between the cannabinoid and opioid systems in this region.