Short Communication: The effect of in ovo creatine monohydrate on early pen use activity in chicks.

First-week survival and egg hatchability are lower in chicks from younger broiler breeder hen flocks. Creatine is a naturally occurring compound synthesised from the amino acid arginine or obtained from the diet and is important in the storage and transport of energy. Previous research found an improvement in the hatch rate but no posthatch performance improvements when fertile eggs from young breeder hens were injected with creatine monohydrate (CrM) on embryonic day 14. This pilot study aimed to further investigate the possibility of early posthatch improvements by examining the activity of chicks during the 1st week posthatch. Behaviours were broadly classified as active or inactive, the pen was split into three areas, and the amount of time spent in the heat lamp, feed hopper, or drinker line areas was recorded. Chicks given in ovo CrM spent less time in the heat lamp area over the whole 7 days compared to saline (t = 2.352, P = 0.021) and control groups (t = 3.336, P = 0.003) and more time in the feed hopper area during the first 4 days compared to the control group (t = 2.174, P = 0.033). This finding suggests that creatine may improve energy reserves in young chicks allowing them to spend more time away from the heat lamp.

Protein instability during HIC: evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond-reduced alpha-lactalbumin variants.

A two-conformation, four-state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four-state model to the effect of salt concentration on the adsorption and unfolding of the model protein alpha-lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild-type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of alpha-lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild-type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (approximately 6-8 kJ/mol) due to increased instability of the protein. Finally, the four-state model is used to accurately predict the apparent adsorption strength of a disulfide bond-reduced variant.

[Calcification in nonfunctioning transplanted kidneys]. / Calcificación del injerto renal no funcionante.

Failed renal allografts often are left in situ in patients who revert to chronic dialysis therapy or who undergo retransplantation. These organs may be the site of massive calcification despite their lack of physiological function. Calcification of an endstage renal allograft is sometimes found incidentally. We report here two patients who developed extensive calcification of the renal graft, one was on chronic hemodialysis and the other had a second renal transplantation with normal renal function. The precise pathogenesis of calcification and the factors which determine its tissue localization are unclear. Factors postulated to promote the development of metastatic calcification include an elevated calcium phosphate product, severe secondary hyperparathyroidism, aluminium toxicity and duration of dialytic therapy. In some cases local factors related with the chronic inflammatory rejection process are probably involved as well. However, the exact relative contribution of these factors remains unresolved. Unless specific clinical indications are present, transplant nephrectomy is not necessary for calcified end-stage renal allografts.

[Hemoperfusion in the treatment of acute valproic acid intoxication]. / Tratamiento con hemoperfusión de la intoxicación aguda por ácido valproico.

Valproic acid is increasingly used in the treatment of epilepsy, and also prescribed for bipolar affective disorders, schizoaffective disorders, schizophrenia and migraine prophylaxis. Valproic acid intoxication with suicide attempt is a relatively common clinical problem that can result in coma, respiratory depression, pancytopenia, hemodynamic instability and death. The drug's relatively low molecular weight, small volume of distribution and saturable protein-binding render it potentially amenable to exracorporeal removal (hemodialysis, hemoperfusion or hemofiltration ), but published experience is scarce. We describe a case report involving valproic acid intoxication with ingestion of ethanol, who was successfully treated with charcoal hemoperfusion. With this treatment the half-life of valproic acid was reduced with rapid lowering of valproic acid levels and clinical improvement. Based on our experience in this patient and a review of previously reported cases, charcoal hemoperfusion should be considered for serious valproic acid intoxication because free as well as bound drug fractions are eliminated via this technique.

[Treatment of acute lithium intoxication with high-flux haemodialysis membranes]. / Tratamiento de la intoxicación aguda por litio mediante hemodiálisis con dializadores de alta eficiencia.

Lithium carbonate is commonly prescribed for the treatment of bipolar (manic-depressive) disorders. However, because of its narrow therapeutic index an excessive elevation of serum lithium concentration, either during chronic maintenance therapy or after an acute overdose, can result in serious toxicity. In addition to supportive care, the established treatment of severe lithium toxicity is haemodialysis. Conventional haemodialysis can reduce serum lithium rapidly, but post-dialysis rebound elevations with recurrent toxicity have been documented in old publications. High-flux membranes should be capable of removing more lithium per hour of haemodialysis, but published values are not available. We report here three patients with acute lithium intoxication who were treated successfully with bicarbonate and high-flux haemodialysis membranes. Our patients presented with a severe degree of intoxication, based on the amount of drug ingested, the initial serum lithium level, the severity of neurologic symptoms and systemic manifestations. Two patients developed acute renal failure probably as a result of volume depletion since it was rapidly reversible by haemodialysis and infusion therapy. In addition, consecutive haemodialysis sessions and improvement of renal function allowed a rapid decrease in serum lithium levels without haemodynamic instability or rebound elevations in lithium concentration. The effectiveness of the procedure in these cases can be attributed to the use of bicarbonate dialysate and high-efficiency dialysers. This is the first report describing the effect of high-efficiency dialysers on lithium pharmacokinetic. Using this technique the elimination rate of lithium was found to be greater than previously reported with haemodialysis.

Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.

Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms.

Crystal structure of Lysbeta(1)82-Lysbeta(2)82 crosslinked hemoglobin: a possible allosteric intermediate.

The crystal structure of human hemoglobin crosslinked between the Lysbeta82 residues has been determined at 2.30 A resolution. The crosslinking reaction was performed under oxy conditions using bis(3, 5-dibromosalicyl) fumarate; the modified hemoglobin has increased oxygen affinity and lacks cooperativity. Since the crystallization occurred under deoxy conditions, the resulting structure displays conformational characteristics of both the (oxy) R and the (deoxy) T-states. beta82XLHbA does not fully reach its T-state conformation due to the presence of the crosslink. The R-state-like characteristics of deoxy beta82XLHbA include the position of the distal Hisbeta63 (E7) residue, indicating a possible reason for the high oxygen affinity of this derivative. Other areas of the molecule, particularly those thought to be important in the allosteric transition, such as Tyrbeta145 (HC2) and the switch region involving Proalpha(1)44 (CD2), Thralpha(1)41 (C6) and Hisbeta(2)97 (FG4), are in intermediate positions between the R and T-states. Thus, the structure may represent a stabilized intermediate in the allosteric transition of hemoglobin.

Detection of Streptococcus mutans by PCR amplification of spaP gene.

Synthetic oligonucleotide primers were used in the polymerase chain reaction (PCR) to amplify a sequence of the spaP gene, which encodes the surface protein antigen I/II of Streptococcus mutans. A DNA fragment of c. 192 bp was amplified from lysed S. mutans cells or isolated DNA. With S. mutans cells, the lower limit of detection was 4-40 cfu. With these primers, 13 reference and 50 clinical strains of S. mutans were identified. Amplification of the 192-bp product was not demonstrated when 41 strains of other streptococcal and non-streptococcal species were tested. The spaP gene PCR has potential for the rapid diagnosis of S. mutans infections.

Modeling NMR lineshapes using logspline density functions.

Distortions in the FID and spin echo due to magnetic field inhomogeneity are proved to have a representation as the characteristic function of some probability distribution. In the special case that the distribution is Cauchy, the model reduces to the conventional Lorentzian model. A more general and flexible representation is presented using the Fourier transform of a logspline density. An algorithm for fitting the model is described, the performance of the model and algorithm is investigated in applications to real and simulated data sets, and the logspline approach is compared to a previous Hermitian spline approach and to the Lorentzian model. The logspline model is more parsimonious than the Hermitian spline model, provides a better fit to real data, and is much less biased than the Lorentzian model.

Inhibition of human platelet aggregation by gangliosides.

The content and composition of gangliosides is modified upon platelet stimulation, suggesting that these lipids may play functional roles in platelet physiology. Therefore, the effect of exogenously added gangliosides on human platelet aggregation was evaluated. The pretreatment of platelets with a mixture of total gangliosides from bovine brain and a series of purified mono-, di- and tri-sialogangliosides partially inhibit the collagen-induced aggregation process and ATP release and completely block the generation of the second aggregation wave when ADP is used as agonist. The inhibition was exerted at around 100 microM by G(TOT) as well as purified G(M1), G(M3), G(D1a), and G(T1b) gangliosides, whereas asialoG(M1) and sulphatide did not show a significant influence on platelet aggregation. Thrombin, Ca(2+) ionophores (A23187 and Ionomycin), arachidonic acid, and U46619 were unable to bypass the inhibitory effect exerted by gangliosides, suggesting that gangliosides inhibit platelet aggregation by inhibiting the synthesis or action of prostaglandins. Gangliosides inhibited U46619-induced aggregation, thus suggesting that they block the action of thromboxane A(2). Epinephrine induces a partial aggregation on gangliosides-treated platelets, similar to fluoroaluminate and phorbol myristate acetate, indicating that these platelets are still functional. To summarize, these results indicate that the major pathway(s), but not all, driving to the aggregation process following the interaction of ligand-receptor may be blocked by pretreatment of human platelets with gangliosides.

Structural studies of chemokines that inhibit HIV-1 entry.

The chemokine receptors CCR5 and CXCR4 have emerged as essential mediators of HIV-1 pathophysiology, functioning as co-receptors for viral entry into cells. The physiological agonists of these receptors inhibit HIV-1 infection in vitro. The discovery of small molecules that disrupt the interactions between HIV-1 and chemokine receptors is one strategy to limit the spread of the virus. These compounds will complement already existing therapies that include HIV-1 reverse transcriptase and protease inhibitors. The complete structural elucidation of a chemokine ligand-receptor complex would be valuable for rational drug design, but has yet to be achieved. Structural studies of chemokine agonists and antagonists can also be useful in understanding interactions that may be important for drug optimization. This review examines the surface properties of the chemokine ligands human SDF-1alpha and HHV-8 vMIP-II, with a goal of determining receptor-interacting sites. In combination with site-directed mutagenesis of the chemokines and structure-activity relationships of chemokine-based peptides, this approach will lead to a better understanding of the interactions in the chemokine ligand-receptor system.

Mapping of lactate and N-acetyl-L-aspartate predicts infarction during acute focal ischemia: in vivo 1H magnetic resonance spectroscopy in rats.

The time course, anatomic distribution, and extent of changes in cerebral lactate, N-acetyl-L-aspartate (NAA), and other metabolite levels determined by three-dimensional in vivo 1H magnetic resonance spectroscopy and single-voxel spectral analysis after middle cerebral artery occlusion in rats. Increased lactate was detected in the central ischemic region within 1.3 hours after the onset of permanent occlusion (n = 22) or 0.5 hour after the onset of 1 hour of temporary occlusion and then reperfusion (n = 8). Permanent occlusion resulted in persistent lactate elevation and a 25.4 +/- 4.1% reduction in the NAA peak after 1.3 hours; NAA was almost completely depleted after 24 hours. Results also demonstrated delayed depletion of all other magnetic resonance spectroscopy-visible 1H metabolites, including creatine, choline, and glutamate, after permanent occlusion. After 1 hour of temporary focal ischemia, lactate returned to nearly normal levels within 0.4 hour after the onset of reperfusion; at 72 hours, a recurrent increase in lactate and a new decrease in NAA were observed, suggesting delayed tissue injury. Histological analysis, performed in 10 rats, demonstrated infarcts that corresponded in distribution to regions of NAA depletion at 72 hours. These findings indicate that lactate elevation is a sensitive early marker of ischemia; however, temporary recovery of lactate accumulation after reperfusion did not predict sustained metabolic recovery. In contrast, NAA depletion within 1.3 hours after the onset of ischemia identified central ischemic regions that were destined for infarction. Potential clinical applications include selection and monitoring of therapeutic intervention, as well as prediction of outcome, in patients with acute stroke.

In situ analysis of protein chromatography and column efficiency using magnetic resonance imaging.

Magnetic resonance imaging has been used to visualize size-based protein separations inside operating chromatography columns. The effects of flow nonuniformity have been observed and analyzed quantitatively through concentration profiles of tracers measured inside the column. Analysis of these profiles provides local and averaged intracolumn plate height values for characterization of dispersion and flow nonuniformity. The magnetic resonance measurements compare favorably with conventional chromatographic measurements of column efficiency and provide more detailed insights into nonideal column performance.

Unfolding and conformational distributions during protein precipitation.

The association of misfolded proteins, or aggregation, is a critical problem in a number of human diseases as well during the expression, refolding, formulation, and delivery of therapeutic proteins. In this study, we investigate lysozyme precipitation with hydrogen exhange using nuclear magnetic resonance (NMR) and mass spectrometry (MS). We show that MS can reveal the presence of conformational distributions, albeit without the detailed structural information afforded by NMR. Further, we find that increases in precipitant concentration alter the structure and composition of precipitates. The selective unfolding of one portion of the protein in these precipitates is correlated with hydrogen exchange patterns observed under nonprecipitating conditions and in other studies of lysozyme.

Mechanical deformation of compressible chromatographic columns.

A one-dimensional model of mechanical deformation of compressible chromatography columns is presented. The model is based on linear elasticity and continuum mechanics and is compared to a more complete two-dimensional model and one-dimensional porosity profiles measured by NMR imaging methods. The model provides a quantitative description of compression and the effects of wall support during scale-up. A simple criterion for the significance of wall support as a function of both diameter and length is also developed. Although the model accounts only for mechanical deformation, flow compression can be included, and validation presented here suggests that a more complete model may be valuable for anticipating the effects of scale and aspect ratio on pressure-flow behavior of compressible columns.

Three dimensional outer volume suppression for short echo time in vivo 1H spectroscopic imaging in rat brain.

Three-dimensional in vivo spectroscopic imaging in rat brain requires additional localization to reduce the effects of lipid contamination. Outer volume suppression has been shown to be effective in two dimensions. We have extended this approach to three dimensions with additional outer volume suppression conforming to the shape of the rat brain. The technique suppresses the pericranial lipid effectively, allowing sampling of volumes close to the skull and effective localized shimming. These benefits permit three-dimensional 1H magnetic resonance spectroscopic imaging to be performed over most of the brain at shorter echo times, providing spectra more amenable to quantitative analysis.

Three-dimensional 1H spectroscopic imaging of cerebral metabolites in the rat using surface coils.

Three dimensional metabolite maps of protonated metabolites were obtained using 1H magnetic resonance spectroscopic imaging at 7 T. Surface coils were used to increase sensitivity and spatial resolution significantly over a volume coil two-dimensional acquisition. Adiabatic pulses were employed to provide homogeneous B1 excitation and frequency selective refocusing over the volume of the rat brain. These techniques were employed to obtain three-dimensional spectroscopic imaging spectra from nominal voxel volumes of 9-30 microliters from rat brain. The improved spatial resolution and sensitivity are also demonstrated with studies of focal ischemia in the rat.

Detection of Fusarium oxysporum f. sp. melonis Race 1 in Soil in Colima State, Mexico.

During the winters of 2002 and 2003, a wilt occurred in melons cultivated on 1,500 ha in Colima State, Mexico. Yield losses reached 25% of final production, despite soil disinfestation with 60% methyl bromide and 40% chloropicrin. On the basis of the observation of plants with necrotic xylem, yellowing, and wilting of leaves, this disease was identified provisionally as Fusarium wilt. During February 2003, four soil samples from affected fields were plated onto a Fusarium-selective medium (1), which resulted in the detection of 2,260 ± 357, 179 ± 76, 668 ± 357, and 1,391 ± 256 CFU/g of F. oxysporum (3). Thirty-one randomly chosen isolates were used to inoculate differential cultivars of melon as described by Risser et al. (4). The cultivars were Amarillo Canario (susceptible to all races), Diana (resistant to races 0 and 2), Tango (resistant to races 0 and 1), and Vulcano (resistant to races 0, 1, and 2) (2). Ten plants of each cultivar, grown on sterilized vermiculite, were inoculated at the first true-leaf stage by drenching with 200 ml of a conidial suspension (1 × 105 CFU/ml) of each isolate. Noninoculated plants of each cultivar served as controls. Plants were maintained in a growth chamber with a 16-h photoperiod (18 × 103 lux) and temperatures at 23 to 25°C. Yellowing, wilt, and vascular discoloration symptoms developed on cvs. Amarillo Canario and Diana following inoculation with each of the 31 isolates, while noninoculated plants remained symptomless. F. oxysporum was consistently reisolated on potato dextrose agar from the affected plants. On the basis of the combination of affected cultivars, all isolates were identified as F. oxysporum f. sp. melonis race 1. To our knowledge, this is the first report of F. oxysporum f. sp. melonis race 1 in Colima State, Mexico. References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) J. Marín Rodríquez. Portagrano 2004. Vadmecum de Variedades Hortícolas. Agrobook, Spain. 2004. (3) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983. (4) G. Risser et al. Phytopathology 66:1105, 1976.

[Pulmonary valvuloplasty in children with cyanotic cardiopathy]. / Valvuloplastia pulmonar en niños con cardiopatías cianóticas.

We present 5 patients with cyanotic congenital heart disease in whom a pulmonary valvuloplasty was performed as palliative therapy. The patients symptoms were hypoxemic spells, very low oxygen saturation and no weight gain. The oxygenation improved and symptoms disappeared. We believe that in properly selected patients this technique represents a good therapeutic modality.