Evaluación de algunos factores de riesgo para la presentación de síndrome de úlcera gástrica (SUGE) en el caballo criollo colombiano en el Valle de Aburrá, Antioquia (Colombia) / Evaluation of some risk factors for the presentation of equine gastric ulcer syndrome (EGUS) in colombian creole horse in Valle de Aburrá, Antioquia (Colombia)

RESUMEN Las úlceras gástricas se definen como la pérdida de continuidad de la mucosa gástrica. Se originan principalmente por desequilibrio entre factores injuriantes y de protección del estómago. Generalmente se manifiestan con signos como pérdida de peso, cólico, bruxismo e inapetencia, conllevando al bajo rendimiento del animal y pérdidas económicas. El objetivo del presente estudio fue determinar la prevalencia y factores de riesgo para la presentación de SUGE en CCC del Valle de Aburra. Se realizó gastroscopia a 100 animales de diferentes pesebreras, entre dos y 25 años de edad; cada estómago se caracterizó macro y microscópicamente. Las lesiones macroscópicas se valoraron de acuerdo con el grado de lesión según el Equine Gastric Ulcer Council, y la clasificación microscopica se basó en una adaptación hecha por patrones de descripción de lesiones más utilizados como: The sidney system y Equine gastritis grading system (EGGS). Para el análisis de los resultados, se tuvieron en cuenta variables como la intensidad del ejercicio, alojamiento, tipo y frecuencia de alimentación, transporte y frecuencia del mismo. El 65% de los equinos evaluados presentaron lesiones compatibles con SUGE. Macroscópicamente el grado 2 de severidad fue el más común (38%), con mayor presentación en la región aglandular. Microscópicamente el edema en la región fundica y pilórica fue la lesión más frecuente (35,2% y 30,7%, respectivamente). Los principales factores de riesgo asociados a la presentación de las lesiones fueron el transporte de los animales y la intensidad del ejercicio.

ABSTRACT Gastric ulcers are defined as the loss of continuity of the gastric mucosal. They are mainly caused by an imbalance between harmful and protective factors of the stomach. It generally manifests itself with signs such as weight loss, colic, bruxism and inappetence, which can lead to low performance and economic losses. The objective of the present study was to determine the prevalence and risk factors for the presentation of SUGE in CCC of the Valle del Aburra. Gastroscopy was made to 100 animals from different stables, between two and 25 years of age; each stomach characterized, macro and microscopically. Lesions are evaluate depending on the degree of injury to the "Equine Gastric Ulcer Council". Microscopic lesions classification was based on an adaptation by patterns most commonly used description of injuries such as: the Sidney System and the equine gastritis grading system (EGGS). For the analysis of the results, variables such as exercise intensity, accommodation, type and frequency of feeding, transportation and its frequency were taken into account. 65% of the equines evaluated presented SUGE-compatible lesions, of which the most common degree of severity macroscopically was grade 2 (38%), with grater presentation in the non-glandular region. Microscopically, edema in the fundic and pyloric region was the most frequent (35.2% and 30.7%, respectively). The main risk factors associated with the presentation of the lesions were the transport of the animals and the intensity of the exercise.

Morphological characterisation of the coronary arteries in African sheep (Ovis orientalis). Differential analysis with those of humans and other animal species.

BACKGROUND: Despite the importance of the coronary system in the African sheep as a possible experimental model, there is little information about this particular vascular system. The objective of this investigation was to characterise morpho- logically the coronary arteries and their branches in African sheep. MATERIALS AND METHODS: This descriptive cross-sectional study evaluated the coronary arteries and their branches of 62 hearts of short hair sheep. The right and left coronary ostia were perfused with a semi-synthetic resin (Palatal GP40L 85%; styrene 15%) dyed with mineral red. The morphological characteristics were evaluated using a digital calibrator and the biometrics of the coronary arteries and their branches were registered. RESULTS: The right coronary artery had a proximal calibre of 2.11 ± 0.46 mm. The subsinusal interventricular branch ended at the middle third of the homonym sulcus in 19 (30.6%) specimens. The left coronary artery had a diameter of 5.38 ± ± 1.59 mm and a length of 4.67 ± 3.32 mm. This artery bifurcated itself in the paraconal interventricular branch and the left circumflex branch in 57 (91.9%) hearts and trifurcated with an additional left diagonal branch in 5 (8.1%) spec- imens. Left coronary artery dominance was observed in 51 (82.3%) specimens, whereas in 11 (17.7%) cases the coronary circulation dominance was balanced. CONCLUSIONS: Due to the similitude in the features of the coronary arteries between African sheep and humans, this animal model can be proposed for procedural and haemodynamic activities.

Identification and characterization of two variants of the Hfq-sRNA-chaperone in the fish pathogen Piscirickettsia salmonis.

Small RNA and chaperone proteins form synergistic duos that play pivotal roles in controlling gene expression in bacteria. This is the case for Hfq, a highly pleiotropic pretranslational modulator of general protein expression, which responds to harsh environmental conditions and influences fitness and virulence in a wide range of pathogenic Enterobacteria. Given this relevancy, we evaluated the presence and potential role of Hfq in the fish pathogen Piscirickettsia salmonis, a Gram-negative bacterium that threatens the sustainability of Chilean salmon production. Using bioinformatics tools were identified and characterized two variants of Hfq, which share the consensus RNA-binding domains and the active sites described functional Hfq other bacteria. Additionally, we demonstrated that hfq-1 and hfq-2 were transcriptionally active when growing in cell-free media and in infected susceptible fish cell line. Expression of both genes differed under different growth conditions and under stress, suggesting that their roles might be independent and different, depending on the bacterial physiological status. In conclusion, we demonstrate the existence of two different and functional ORF coding for the hfq marker in marine bacteria and a preliminary analysis indicating that these two novel proteins might have relevant roles in the biology and pathogenic potential of P. salmonis.

Anatomical study of the renal excretory system in pigs. A review of its characteristics as compared to its human counterpart.

BACKGROUND: Despite the importance of the pyelocalyceal system in the pig as an experimental model, there is little information about this particular anatomical subject. We determined the morphological characteristics of the renal excretory system in pigs. MATERIALS AND METHODS: This descriptive cross-sectional study evaluated 130 pairs of kidneys of pigs destined to slaughter. The pyelocalyceal system was subjected to injection technique - corrosion by infusion of polyester resin (85% Palatal and 15% Styrene) and subsequent infusion in potassium hydroxide (KOH) for 10 days. The significance level used was p < 0.05. RESULTS: The renal excretory system is characterised by the presence of type A major cranial and caudal calyxes seen in 34.3% of the kidneys (type A1 in 30% and type A2 in 4.3%). type B calyxes, corresponding to minor calyxes draining directly into the renal pelvis, were present in 65.7% of the specimens (type B1 59.2%; type B2 6.5% of the cases). The number of minor calyxes in the collector system was 7.9 ± 2.27 with statistically significant differences in side (p = 0.0047). CONCLUSIONS: The morphometric characteristics of the kidneys in this study are slightly smaller than reported in humans. Similarly, the incidence of type A renal excretory system distribution is highest in humans and lowest in pigs. Due to its few morphological differences, the pig kidney is an excellent model for teaching- -learning processes, for research purposes, and for training of urologic applications.

Evaluation of coronary dominance in pigs; a comparative study with findings in human hearts / Avaliação da dominância coronária em suínos. Estudo comparativo com os achados em corações humanos

Coronary dominance in swine has been poorly evaluated. The frequencies of each type of dominance have been described, but few details have been given as to the different expressions of each one. The aim of this study was to characterize coronary dominance in commercial breed swine. One hundred and fifty eight pig hearts were evaluated. The coronary arteries (CA) were infused with synthetic resin (Palatal 85% and Styrene15%) through the ostia after channeling. The coronary artery that gives origin to the posterior interventricular artery (PIA), and the site of termination of both the circumflex arteries (CXA), and left retroventricular branch (LRVB) were determined in order to establish the coronary dominance pattern. Right coronary dominance was found in 105 hearts (66.5%), and a balanced circulation in 53 specimens (33.5%). No dominance was observed for the left coronary artery in the hearts studied. The CXA ended on the posterior aspect of the left ventricle in 101 samples (64%) and on the crux cordis in 55 specimens (34.8%). In two specimens (1.3%) it ended as a left marginal artery. In all cases the PIA was a branch of the RCA, and was long in 105 hearts (66%), 55% of which corresponded to males and 45% to females, but this difference was not statistically significant (p=0.77). The AIA ended on the apex in 126 specimens (80%), 71 of which (56%) corresponded to males and 55 (44%) to females (p=0.74)...

A dominância coronária em suínos tem sido pouco avaliada. Descreveram-se as frequências de cada um dos tipos, mas não detalharam as diferentes expressões de cada um deles. O objetivo deste estudo foi caracterizar a dominância coronária em suínos de raças comerciais. Avaliaram-se 158 corações de suínos. As artérias coronárias (AC) foram infundidas através da canalização dos seus ostium com resina sintética (Palatal 85% e Estireno15%). Para estabelecer o tipo de dominância coronária, determinou-se de qual coronária desprendia-se a artéria interventricular posterior (AIP) e o lugar de finalização das artérias circunflexa (ACX) e do ramo retro ventricular esquerdo (RRVI). Encontrou-se dominância coronária direita em 105 corações (66%) e circulação balanceada em 53 exemplares (34%). Não foi observada a dominância coronária esquerda nos corações estudados. O calibre proximal e médio da ACD nos casos de dominância coronária direita foi de 3,84 ± 0,80 mm. Por outro lado, encontrou-se que este mesmo calibre nos corações com dominância coronária balanceada foi de 3,97 + 0,79 mm. (p=0,88). A ACX finalizou-se na face posterior do ventrículo esquerdo em 101 amostras (64%) e na crux cordis em 55 exemplares (34,8%). A AIP emergiu em todos os casos da ACD sendo comprida em 105 corações (66%) dos quais 55% correspondia a machos e 45% a fêmeas, sem que esta diferença fosse estatisticamente significativa (p=0,77). A AIA finalizou-se no ápice em 126 exemplares (80%), dos quais 71 (56%) corresponderam a machos e 55 (44%) a fêmeas (p=0,74)...

Characterisation of myocardial bridges in pigs: a comparative anatomical analysis with the human heart.

BACKGROUND: Few studies have been conducted in pigs concerning the presence of myocardial bridges (MB) on the coronary arteries and their branches, and some of them have evaluated small samples. The objective of this study was to characterise MB in pigs of commercial breeds. MATERIALS AND METHODS: One hundred and fifty eight hearts of pigs destined to the slaughterhouse with stunning method were studied. The coronary arteries were perfused with polyester resin (palatal 85% and styrene 15%) and then subjected to potassium hydroxide infusion to remove the subepicardial fat. RESULTS: Ninety three MB were found in 67 (42.4%) specimens, 43 (46%) of which were located on branches of the right coronary artery, 38 (41%) on branches of the left coronary artery and 12 (13%) on both vessels. The MB occurred in 26 (38.8%) females and 41 (61.2%) males, but the difference was not statistically significant (p = 0.23). Single MB were most common (70%), followed by the presence of 2 (21%) MB in different vessels. the subsinusal interventricular artery was the vascular structure with the largest number of MB (46.2%), with its middle third being the most compromised segment (79%). The mean length of the MB was 11.23 ± 5.67 mm and the thickness of the suprapontine myocardium was 1.13 ± 0.48 mm. CONCLUSIONS: The frequency, localisation, and length of the MB reported in pigs are consistent with the findings of the present study, whereas in humans the MB involve mainly the anterior interventricular artery and are longer.

Anatomic study of the right coronary artery in pigs: feature review in comparison with the human artery / Estudio anatómico de la arteria coronaria derecha en cerdos: revisión de sus características en comparación con la del humano

A few studies conducted on the morphology of coronary arteries in pigs emphasize the description of its course and number of branches. The objective of this study was to determine the anatomic expression of the right coronary artery (RCA) in pigs of commercial stock. One hundred and fifty eight hearts obtained from pigs destined to sacrifice were studied. The RCA were perfused with polyester resin (Palatal 85 percent and Styrene 15 percent) and then subjected to KOH infusion for 5 minutes to release the subepicardial fat. Diameters were measured and courses were evaluated. Results: The diameter of the RCA in its proximal portion was 3.85 mm (SD 0.75). The right artery of the cone originated from the RCA in 100 specimens (63.3 percent) and from the aorta (third coronary) in 8 specimens (5.1 percent). The posterior interventricular branch (PIB) reached the apex in the majority of the cases (49.3 percent). Its proximal diameter was 2.75 mm (SD 0.61). The sinoatrial node branch (SAB) and the atrioventricular node branch (AVNB) originated from the RCA in 100 percent of the specimens. The diameters of the SAB and AVNB were 1.1 mm (SD 0.30) and 1.16 mm (SD 0.30), respectively. The posterior right diagonal branch was not observed. There is an agreement with most prior studies with respect to both SAB and AVNB emerging from the RCA. Morphometric and qualitative knowledge of the RCA and its branches in pigs is relevant for the design of procedural and hemodynamic models.

Los escasos estudios realizados con relación a la morfología de las arterias coronarias en porcinos enfatizan en descripciones de sus trayectorias y número de ramas. El objetivo del estudio fue determinar la expresión anatómica de la arteria coronaria derecha (ACD) en cerdos de razas comerciales. Fueron estudiados 158 corazones extraídos de cerdos destinados al sacrificio. Las ACD fueron perfundidas con resina poliéster (Palatal 85 por ciento y Estireno15 por ciento) y posteriormente sometidas a infusión de KOH durante 5 minutos para liberar la grasa subepicárdica. Se midieron calibres y evaluaron trayectorias. El calibre de la ACD en su porción proximal fue 3,85 mm (DE 0,75). La arteria derecha del cono se originó de la ACD en 100 especímenes (92,6 por ciento) y de la aorta (tercera coronaria) en 8 (7,4 por ciento). La arteria interventricular posterior (AIP) alcanzó el ápex en la mayoría de los casos (49,3 por ciento.) Su calibre proximal fue 2,75 mm (DE 0,61). La rama del nodo sinusal (RNS) y la rama del nodo atrio ventricular (RNAV) se originaron de la ACD en el 100 por ciento de los especímenes. Los calibres de la RNS y RNAV fueron 1,1 mm (DE 0,30) y 1,16 mm (DE 0,30) respectivamente. No se observó rama posterolateral. Existe concordancia con la mayoría de estudios previos con relación a la emergencia desde la ACD de las ramas RNS y RNAV. El conocimiento morfométrico y cualitativo de la ACD y sus ramas en porcinos, es relevante para el diseño de modelos procedimentales y hemodinámicos.

[Intravenous 3-day Helicobacter pylori eradication therapy is highly effective in patients with bleeding peptic ulcer]. / Alta eficacia erradicadora de la infección por Helicobacter pylori de una pauta terapéutica intravenosa de 3 días de duración en pacientes con úlcera péptica sangrante.

AIMS: To evaluate the efficacy of 3-day intravenous Helicobacter pylori eradication therapy in patients with bleeding peptic ulcer associated with H. pylori infection. MATERIAL AND METHOD: We studied 53 patients admitted to hospital with bleeding of the upper gastrointestinal tract due to peptic ulcer and positive urease test over a 12-month period. After endoscopic diagnosis, intravenous pantoprazole (40 mg/12 hours), metronidazole (500 mg/8 hours) and amoxicillin-clavulanic acid (1,000 mg/200 mg/8 hours) was administered for 72 hours. The efficacy of eradication therapy was evaluated by 13C-urea breath test at least 2 months after the end of treatment. RESULTS: Fifty-one patients were included. Of these, 40 had duodenal ulcer and 11 had gastric ulcer. H. pylori eradication was achieved in 87.5% of those with duodenal ulcers and in 63.6% of those with gastric ulcers (p = 0.066). No adverse reactions or episodes of bleeding recurrence were found and none of the patients withdrew from treatment. CONCLUSIONS: The ultra-short course eradication therapy used in this study is highly effective. Its efficacy is similar to that of oral treatment and it avoids certain problems such as adverse effects and adherence to treatment.

On-column ligand synthesis coupled to partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to a receptor.

This paper describes a two-step procedure whereby on-column ligand synthesis and partial-filling affinity capillary electrophoresis (PFACE) are sequentially coupled to each other to determine the binding constants of 9-fluorenylmethoxy carbonyl (Fmoc)-amino acid-D-Ala-D-Ala species to vancomycin (Van) from Streptomyces orientalis. In this technique four separate plugs of sample are injected onto the capillary column and electrophoresed. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and running buffer, respectively. The fourth sample plug contains an increasing concentration of Van partially-filled onto the capillary column. Upon electrophoresis the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the plugs of Van and Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Analysis of the change in the relative migration time ratio of the Fmoc-amino acid-D-Ala-D-Ala peptide relative to the non-interacting standards, as a function of the concentration of Van, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques.

Multiple-step ligand injection affinity capillary electrophoresis for determining binding constants of ligands to receptors.

This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.

On-column derivatization and analysis of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis.

This work demonstrates the use of an in-capillary procedure for derivatization of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis (CE). Migrating in an uncoated fused-silica capillary, plugs of substrate and anhydride are injected separately and electrophoresed. Differential transport velocities permit the separate zones to penetrate each other under an applied field, thereby facilitating reaction. In initial experiments the extent of reaction between tryptophan and acetic anhydride was examined and product amounts quantitated by CE. In separate experiments a series of amino acids and peptides were injected into the capillary and reacted with phthalic anhydride on-column to yield the phthalic derivatized species. Finally, on-column derivatization of alkylamines with phthalic anhydride was investigated and electrophoretic mobility related to molecular weight of the derivatized amines. These procedures illustrate the use of the capillary as a microreactor in the facile synthesis of derivatized molecules and ease of quantitation of reaction products under conditions of electrophoresis.

Use of capillary electrophoresis and indirect detection to quantitate in-capillary enzyme-catalyzed microreactions.

The use of capillary electrophoresis and indirect detection to quantify reaction products of in-capillary enzyme-catalyzed microreactions is described. Migrating in a capillary under conditions of electrophoresis, plugs of enzyme and substrate are injected and allowed to react. Capillary electrophoresis is subsequently used to measure the extent of reaction. This technique is demonstrated using two model systems: the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13), and the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose-1,6-bisphospatase (FBPase, EC 3.1.3.11). These procedures expand the use of the capillary as a microreactor and offer a new approach to analyzing enzyme-mediated reactions.

Estimation of receptor-ligand interactions by the use of a two-marker system in affinity capillary electrophoresis.

The study of receptor-ligand interactions by affinity capillary electrophoresis (ACE) requires an accurate form of analysis. Here, we examine the use of two noninteracting standards (markers) in the analysis of binding constant data in ACE studies. This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and the dipeptide N-acetyl-d-Ala-d-Ala. In this procedure a plug of receptor and noninteracting standards is injected, and analysis of the change in the relative migration time ratio of the receptor, relative to the noninteracting standards, as a function of the concentration of the ligand yields a value for the binding constant. The findings described here demonstrate that data from ACE studies can best be analyzed using two noninteracting standards, yielding values comparable to those estimated using other binding and ACE techniques.

Use of a partial-filling technique in affinity capillary electrophoresis for determining binding constants of ligands to receptors.

This work evaluates the concept of a partial-filling technique in affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique the capillary is first partially-filled with ligand followed by a sample of receptor and non-interacting standard and electrophoresed. Analysis of the change in the mobility ratio, M, of the receptor, relative to the non-interacting standard, as a function of the concentration of the ligand, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.

Use of mobility ratios to estimate binding constants of ligands to proteins in affinity capillary electrophoresis.

This work evaluates the use of mobility ratios (M) to estimate binding constants of proteins to ligands using affinity capillary electrophoresis (ACE). This concept is demonstrated using two model systems: vancomycin (Van) from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). A plot of change in M (deltaM) over the concentration of ligand [L] versus deltaM yields a more useful representation of the Scatchard plot in capillary electrophoresis (CE) than traditional plots of the change in mobility delta mu over [L] versus delta mu in a wide set of circumstances, especially when comparing electropherograms obtained in the presence of substantial variations in electroosmotic flow. Altering the voltage and/or capillary length of the CE system produced only small variations in M, but much larger changes in the more standard measures of migration used by the mu form of analysis. The use of M in the Scatchard analysis offers a new approach to estimating binding constants of ligands to proteins using ACE.

Double enzyme-catalyzed microreactors using capillary electrophoresis.

This work evaluates the concept of a double enzyme-catalyzed microreactor using capillary electrophoresis (CE). Migrating in a capillary under electrophoresis conditions, plugs of substrate and two enzymes are injected separately in buffer and allowed to react. Extent of reaction and product ratios were subsequently determined by CE. This concept is demonstrated using two model systems: the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) by hexokinase (HK, EC 2.7.1.1) and apyrase (APY, EC 3.6.1.5), respectively, in the conversion of glucose to glucose-6-phosphate and inorganic phosphate, respectively, and the conversion of nicotinamide adenine dinucleotide, reduced form (NADH), to nicotinamide adenine dinucleotide (NAD) and back to NADH by lactate dehydrogenase (LDH, EC 1.1.1.27) and glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), respectively, in the conversion of pyruvate to lactate and glucose-6-phosphate (glc-6-P) to 6-phosphogluconate, respectively. These procedures illustrate the use of the capillary as a double microreactor and the ease of quantitation of reaction products under conditions of electrophoresis.

Carboracycles: Macrocyclic Compounds Composed of Carborane Icosahedra Linked by Organic Bridging Groups.

A family of macrocyclic compounds are described, together with their precursors. These cycles are composed of icosahedral carboranes linked via their carbon vertices through 1,3-trimethylene, alpha,alpha'-1,3-xylylene, or alpha,alpha'-2,6-lutidylene groups. The compounds cyclo-(1,3-trimethylene-1',2'-closo-1',2'-C(2)B(10)H(10))(4) (6a), cyclo-(1,3-trimethylene-1',2'-closo-9',12'-dimethyl-1',2'-C(2)B(10)H(8))(4) (6b), cyclo-(1,3-trimethylene-1',2'-closo-1',2'-C(2)B(10)H(10))(3) (9), cyclo-(alpha,alpha'-1,3-xylylene-1',2'-closo-1',2'-C(2)B(10)H(10))(2) (11a), cyclo-(alpha,alpha'-1,3-xylylene-1',7'-closo-1',7'-C(2)B(10)H(10))(2) (11b), cyclo-(alpha,alpha'-1,3-xylylene-1',7'-closo-9',10'-dimethyl-1,7-C(2)B(10)H(8))(2) (11c), cyclo-(alpha,alpha'-1,3-xylylene-1',2'-closo-1',2'-C(2)B(10)H(10))(4) (12), cyclo-(alpha,alpha'-1,3-xylylene-1',7'-closo-1',7'-C(2)B(10)H(10))(3) (13), cyclo-(alpha,alpha'-2,6-lutidylene-1',7'-closo-1',7'-C(2)B(10)H(10))(2) (19), and cyclo-(alpha,alpha'-2,6-lutidylene N-oxide-1',7'-closo-1',7'-C(2)B(10)H(10))(2) (20) have been synthesized. The structures of 6a, 6b, 9, 11a, 11b, 11c, 12, and 19 have been determined by X-ray crystallography. Crystal data: for 6a, triclinic, space group P&onemacr;, a = 11.131(2) Å, b = 12.642(2) Å, c = 12.996(2) Å, alpha = 84.383(6) degrees, beta = 65.884(6) degrees, gamma = 97.292(5) degrees, Z = 1, R = 0.079; for 6b, monoclinic, space group P2(1)/a, a = 13.500(2) Å, b = 31.141(3) Å, c = 13.831(2) Å, beta = 99.90(1) degrees, Z = 2, R = 0.097; for 11a, monoclinic, space group C2/c, a = 14.5682(8) Å, b = 14.5046(8) Å, c = 16.1998(8) Å, beta = 95.631(2) degrees, Z = 4, R = 0.081; for 11b, monoclinic, space group P2(1)/n, a = 11.650(2) Å, b = 10.606(2) Å, c = 11.730(2) Å, beta = 104.951(6) degrees, Z = 2, R = 0.069; for 11c, orthorhombic, space group Pbca, a = 12.532(2) Å, b = 14.271(2) Å, c = 18.143(3) Å, Z = 4, R = 0.076; for 19, orthorhombic, space group Pcab (No. 61, standard setting Pbca), a = 11.0428(6) Å, b = 11.3785(6) Å, c = 22.533(1) Å, Z = 4, R = 0.074.

Determination of the binding of ligands containing the N-2,4-dinitrophenyl group to bivalent monoclonal rat anti-DNP antibody using affinity capillary electrophoresis.

Affinity capillary electrophoresis has been used to determine the two dissociation constants of the complex between anti-DNP rat monoclonal IgG2b antibody and charged ligands that contained a N-dinitrophenyl group. Singly and multiply charged ligands were used to establish the influence of the charge on the mobility of the complex between Ig and its ligand(s). Zwitterionic buffer additives lessened adsorption of protein to the walls of the capillary. A form of analysis of the binding data is derived that is more useful than Scatchard analysis for certain multivalent systems where cooperativity of binding is in question, but where it is also possible to make plausible assumptions about electrophoretic mobilities of protein and protein-ligand complexes. The uncertainties and assumptions of this analysis are contrasted with those of Scatchard analysis. For this antibody and these monovalent ligands, the dissociation of the ligands from the antibody occurred noncooperatively. The charge on IgG2b at pH 8.3 is estimated to be -8.0 +/- 0.2; this value is obtained by analysis of the electrophoretic mobilities of complexes IgG2bL2, where the ligands L are structurally similar but have different charges (the charges on the ligands were also determined by CE).

Using capillary electrophoresis to follow the acetylation of the amino groups of insulin and to estimate their basicities.

Capillary electrophoresis (CE) is an analytical method that is useful for investigating processes that modify the charge of proteins. This paper explores the ability of CE to rationalize charges and electrophoretic mobilities of a simple protein--insulin and its acylated derivatives--as a function of pH. Insulin is a peptide hormone (MW = 5700) that has two alpha-amino groups (G alpha and F alpha) and one epsilon-amino group (K epsilon). Treatment of insulin with acetic anhydride affords seven derivatives that differ in the sites of acetylation of the three amino groups. Analysis of the pH dependence of the electrophoretic mobilities of these derivatives gives pKa values for the two N-terminal ammonium groups: pKa (G alpha) = 8.4; pKa (F alpha) = 7.1. Values of the total charge of insulin estimated from electrophoretic mobility differ from those estimated from values of pKa for its ionizable groups by less than 0.5 unit for both bovine and human insulins over the range of pH from 5.5 to 9.5. Analysis of the concentration dependence of the electrophoretic mobility of insulin yields a lower limit for the association constant for dimerization of insulin of KD > or = 6 x 10(3) M-1 (25 mM tris and 192 mM Gly, pH 8.4). Studies of electrophoretic mobility as a function of pH and extent of acetylation of amino groups rationalize the charge of insulin in detail. The sensitivity of CE to charge permits the quantitative study of electrostatic properties of proteins in solution. Insulin is a useful small-protein model with which to investigate phenomena in electrophoresis.