Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Kidney Stones

The prevalence of kidney stones has steadily risen during this century; passage of a calculus and a positive family history increase the probability of recurrence. Findings from recent studies on the cause of renal calculi have stressed crystallization and crystal aggregation of stone minerals from supersaturated urine, rather than excessive organic matrix. Absence of normal urine inhibitors of calcium salts is also stressed. Formation of calcium oxalate stones is the major problem. Therapy with decreased calcium and oxalate intake, thiazides, phosphate salts and allopurinol in various combinations has substantially decreased the prevalence of recurrent stones. The rationale for the use of allopurinol is that uric acid salts enhance the tendency for calcium oxalate to crystallize from supersaturated urine. The hypercalciuria seen in 30 percent to 40 percent of patients with oxalate stones is usually caused by intestinal hyperabsorption of calcium. Although patients with uric acid calculi constitute only a small fraction of those in whom stones form, they represent a group in whom good medical therapy, based on sound physiologic principles, has proved extremely successful. Renal tubular syndromes lead to nephrocalcinosis and lithiasis through hypercalciuria, alkaline urine and hypocitraturia, the latter an inhibitor of calcium salt precipitation. Recent advances in surgical techniques are discussed, including the rationale for removing staghorn calculi. The ileal ureter and coagulum pyelolithotomy deserve special emphasis.     

Characterization of cold-sensitive secY mutants of Escherichia coli.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Molecular genetics of septo-optic dysplasia

Septo-optic dysplasia (SOD) is a highly variable condition characterized by midline neurological abnormalities associated with pituitary hypoplasia and optic nerve hypoplasia. The aetiology is unknown. Mutant mice, in which a novel homeobox gene, Hesx1, has been disrupted, exhibit a phenotype that resembles the phenotype of SOD. We therefore wished to explore the possibility that this gene is implicated in SOD. We cloned and sequenced the human homologue HESX1 and screened for mutations in affected individuals using single-stranded conformational polymorphism analysis, followed by cloning and sequencing of any exons which showed a band shift. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain, leading to a loss of in vitro DNA binding. Subsequently, we have identified heterozygous mutations in HESX1 that are associated with milder pituitary phenotypes. Our studies indicate a vital role for Hesx1/HESX1 in forebrain and pituitary development in mouse and man, and hence in some cases of SOD.     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.