Effects of adenosine deaminase on the sensitivity of glucose transport, glycolysis and glycogen synthesis to insulin in muscles of the rat

1. Soleus, extensor digitorum longus (EDL) or hemi-diaphragm muscles of the rat were incubated in the presence of insulin and rates of the processes of glycolysis and glycogen synthesis were measured. 2. The concentrations of insulin required to cause half-maximal stimulation of glycolysis in both soleus and EDL preparations were significantly decreased by the presence of adenosine deaminase in the medium. 3. Adenosine deaminase increased the sensitivity of the process of hexose transport to insulin (in an identical manner to the change in sensitivity of glycolysis) in the EDL preparation. 4. None of the adenosine mediated effects on insulin-stimulated rates of glycolysis were observed in the hemi-diaphragm preparation or on the rates of glycogen synthesis in any of the three muscle preparations. 5. Therefore, changes in the adenosine system in skeletal muscle influence insulin sensitivity regardless of fibre type composition of the muscle.     

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.     

Otolith microstructure indicating growth and mortality among plaice, Pleuronectes platessa L., post-larval sub-cohorts

Growth and mortality of post-metamorphosed plaice were studied by means of daily increments in the sagittal otoliths. The Gompertz model was the best fit to length-at-age data and there were no significant differences between length-at-age and back-calculated lengths. The microstructure pattern of the otoliths at metamorphosis was also used to estimate hatching and settlement distributions. Differential growth and mortality occurred among sub-cohorts; growth rates and mortality were higher in fish that settled earlier. In 1986, the best survival was for a sub-cohort settling in late May to early June. In contrast, in the warmer season of 1987, survival was highest for the second and third sub-cohorts settling in late April and mid May.     

The genetic structure and microdistribution of shoals of Phoxinus phoxinus, the European minnow

Efficient foraging and a reduction in predation risk have been proposed as reasons for shoal formation. Some behaviours in cyprinid shoals are at first sight altruistic (e.g. predator inspection behaviour, reactions to alarm substance), such that kin selection may have been involved in their evolution. If shoaling behaviour does evolve through kin selection, then genetic differentiation is expected to be greater between shoals than within shoals. Such a hypothesis was tested here by examining shoal integrity and the relatedness of individuals within and between shoals in the European minnow, Phoxinus phoxinus , using nuclear and mitochondrial DNA markers. The breeding structure of 13 minnow shoals collected from Dorset and North Wales, U.K., was examined using allozymes. Genetic affinity within and between shoals was tested using mitochondrial DNA and multi-locus DNA fingerprinting. Shoals consisted of a random assortment of allozyme genotypes, shoal members did not share the same maternal mtDNA lineages and DNA fingerprint profiles were as varied within shoals as between them. The data indicate that it is unlikely that kin selection occurs in P. phoxinus and there is no apparent relationship between shoaling behaviour and genotype distribution in this species.     

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.     

Prolonged remission maintenance in acute myeloid leukaemia.

Twenty-five patients with acute myeloid leukaemia were treated with three quadruple drug combinations in predetermined rotation: TRAP (thioguanine, daunorubicin, cytarabine, prednisolone); COAP (cyclophosphamide, vincristine, cytarabine, prednisolone); and POMP (prednisolone, vincristine, methotrexate, mercaptopurine). Fifteen patients (60%) achieved complete remission and five (20%) partial remission. For maintenance, five-day courses of drugs were administered every 14 to 21 days and doses were increased to tolerance. The median length of complete remission was 66 weeks. In eight patients remission maintenance treatment was discontinued and some remained in complete remission for over two years. In this series the remission induction rate was comparable with that reported for other regimens and complete remission lasted longer with this intensive maintenance regimen than with others. Nevertheless, the TRAP programme must still be regarded as only palliative treatment for acute myeloid leukaemia.     

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