Modification by site-directed mutagenesis of the specificity of Erythrina corallodendron lectin for galactose derivatives with bulky substituents at C-2.

Examination of the three-dimensional structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin [(1991) Science 254, 862-866], revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134-Trp135, which can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound galactose. Comparison of the primary sequence of ECorL with that of soybean agglutinin, specific for galactose and its C-2 substituted derivatives, and of peanut agglutinin, specific for galactose only, showed that in soybean agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Ser-Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser-Glu- Tyr-Asn. Three mutants of ECorL were therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser-Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the double mutant L2; Y108T. They were expressed in Escherichia coli, as done for recombinant ECorL [(1992) Eur. J. Biochem. 205, 575-581]. The mutants had the same hemagglutinating activity as native or rECorL. Their specificity for galactose, GalNAc and Me beta GalNDns was examined by inhibition of hemagglutination and of the binding of the lectin to immobilized asialofetuin; in addition, their association constants with Me alpha GalNDns and Me beta GalNDns were measured by spectrofluorimetric titration. The results showed that Y108T had essentially similar specificity as the native and recombinant lectins. The affinity of L2 and L2;Y108T for galactose was also the same as ECorL, but they had a lower affinity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less). This appears to be largely due to steric hindrance by the two additional amino acids present in the cavity region in these mutants. Our findings also provide an explanation for the inability of PNA to accommodate C-2-substituted galactose derivatives at its primary subsite.

Synthesis of methyl alpha- and beta-N-dansyl-D-galactosaminides, probes for the combining sites of N-acetyl-D-galactosamine-specific lectins.

The synthesis of the methyl alpha- and beta-N-dansyl-D-galactosaminides is described using methyl alpha,beta-2-azido-2-deoxy-D-galactopyranoside as starting material. This was reduced to the corresponding methyl alpha,beta-2-amino-2-deoxy-D-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl alpha,beta-N-dansyl-D-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl alpha-N-dansyl-D-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with the N-acetyl-D-galactosamine specific lectin from Erythrina corallodendron.

Interaction of hepatic asialoglycoprotein receptor with asialoorosomucoid and galactolyzed lysosomal alpha-glucosidase.

An asialoglycoprotein receptor was isolated from murine liver and purified more than 1600-fold using 2-fold affinity chromatography on asialoorosomucoid-Sepharose. The purified receptor did not interact with 125I-orosomucoid, but bound to 125I-asialoorosomucoid. The binding of the receptor to asialoorosomucoid was saturable. The dissociation constant of the receptor-asialoorosomucoid complex was 0.4 X 10(-9) M. The molecular mass of the receptor, as determined with the use of specific antibodies by the immunoblotting method, was 43 kDa. High concentrations of unlabeled asialoorosomucoid and of n-aminophenyl-beta-D-galactosyl derivatives of bovine serum albumin, ovalbumin and acid alpha-glucosidase from human liver inhibited the binding of the receptor to 125I-asialoorosomucoid almost completely. The binding of the receptor to 125I-galactolyzed alpha-glucosidase was pH-dependent, with the pH optimum at 8.0-9.0. It was shown that, as in the case of 125I-asialoorosomucoid, the binding of the 125I-galactosyl derivative of alpha-glucosidase occurred in the presence of Ca2+ and was inhibited by N-acetylgalactosamine. Glycoproteins containing galactose as a terminal residue inhibited the interaction of the receptor with 125I-galactolyzed alpha-glucosidase. The possibility of directed transport of the galactolyzed alpha-glucosidase derivative into parenchymous liver cells using receptor-mediated endocytosis is discussed.

Human liver lysosomal alpha-glucosidase modified by chemical galactosylation and isolation of specific antibodies.

Human liver acid alpha-glucosidase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was modified with water soluble carbodiimide in the presence of p-aminophenyl-beta-D-galactopyranoside. The incorporation of the aminophenyl derivative of galactose into alpha-glucosidase caused some changes in the physiocochemical properties of the enzyme: a blue shift in the absorption maximum, an alteration of the total electric charge affecting electrophoretic mobility upon polyacrylamide gel electrophoresis, and acquisition of the ability to interact specifically with Ricinus communis agglutinin. At the same time, the 'galactosylated' enzyme possessed high stability and exhibited catalytic activity towards maltose. The Km values of the native and modified enzymes with maltose were 6 and 5 mM, respectively. p-Aminophenyl-beta-D-galactopyranoside residues incorporated in alpha-glucosidase and in other proteins were found to be antigenic determinants to which the pure antibodies were obtained.

Maturation of hypoxic ventilatory response of the newborn lamb.

The ventilatory response to acute hypoxia was studied in unanesthetized newborn lambs before and after carotid body denervation. Animals studied immediately following operative delivery were compared with a second group who were studied in the 2nd wk of postnatal life. Response time was defined as the interval between the decrease in oxygen saturation in the aortic arch and an increase in tidal volume or ventilatory rate. Animals in the older group demonstrated a significantly shorter response time than those studied immediately following delivery (1.8 s vs. 5.7 s). A ventilatory response was also elicited in carotid body-denervated animals in both study groups. Mean response time following denervation was 29.5 s for the younger and 12.5 s for the older animals. The data suggest that the development of a rapid, carotid body-mediated hypoxic ventilatory response occurs gradually during the first days of postnatal life, and that the central hypoxic response of the fetus (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42:630-635, 1977) persists into postnatal life.

Ventilatory response in the fetal lamb following peripheral chemodenervation.

Carotid infusions of sodium cyanide solution and perfusions of hypoxemic or hypercapnic fetal blood were done before and after peripheral chemoreceptor denervation. Step changes in PaO2 ranged from -11 to -22 Torr; step changes in PaCO2 ranged from +17 to +42 Torr. The cyanide dose was 0.2 mg/kg per loop system. Control perfusions consisted of 25 ml of fetal blood without changes in pH and blood gases. A ventilatory response occurred in the majority of all experimental perfusions regardless of innervation status of the peripheral chemoreceptors. No response occurred with control perfusions. There was a marked variability in the time of onset of ventilatory activity with a delay of greater than 10 s occurring following most perfusions. These studies demonstrate that the fetus has an attenuated ventilatory response to chemical stimuli and that hypoxia stimulates ventilation in the absence of peripheral chemoreceptors.

Is continuous transpulmonary pressure better than conventional respiratory management of hyaline membrane disease? A controlled study.

The influence of continuous positive airway pressure (CPAP) and positive end-expiratory pressure (PEEP) on mortality and complication rates in severe hyaline membrane disease (HMD) was evaluated in a randomized, prospective study. Patients were admitted to the study if the Po2 was less than 50 mm Hg with FiO2 greater than 0.6. Twenty-four patients in each of three weight groups were equally divided between treatment and control groups. The treatment regimen included CPAP (6 to 14 cm H2O) for spontaneously breathing patients and PEEP for patients requiring mechanical ventilation for apnea or hypercapnia (Pco2 greater than 65 mm Hg). Control patients received oxygen and were mechanically ventilated if they had apnea, hypercapnia, or Po2 less than 50 mm Hg with FiO2 greater than 0.8. Oxygenation improved after the start of CPAP or PEEP; however, Pco2 rose after CPAP was initiated. There was no significant difference between treatment and control groups in mortality, requirement for mechanical ventilation, or incidence of pulmonary sequelae. The incidence of pulmonary air-leak was increased with Peep. the findings suggest that CPAP and PEEP have not significantly altered the outcome of HMD.

Application of the hot-wire anemometer to respiratory measurements in small animal.

A hot-wire anemometer was evaluated to determine its suitability for measurement of small tidal volumes. Used with a constant background flow of gas, the output of the hot-wire anemometer was linear and independent of respiratory frequency, temperature, and humidity. The change in output with CO2 concentration was negligible within the physiologic range. The use of a background flow eliminates the need for one-way valves, minimizes dead space, and maintains the flow velocity past the hot wire within its range of linear response.