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A J Northrop

Publications and source records attributed to A J Northrop.

17 recordsLinked to original sources
PubMed1995

Brefeldin A and a synthetic peptide to ADP-ribosylation factor (ARF) inhibit regulated exocytosis in melanotrophs.

We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.

ADP-Ribosylation Factors↗
PubMed1993

rab3-peptide stimulates exocytosis from mast cells via a pertussis toxin-sensitive mechanism.

Synthetic peptides of the putative effector domain of members of the rab3 gene family of small GTP-binding proteins have been shown to have potent actions on vesicular transport and exocytosis [1,2]. Here, we use similar rab3-effector domain peptides to study their role in intracellular signalling in mast cells. We find that rab3-like peptides stimulate exocytosis and decrease cyclic 3',5'-AMP levels in these cells when applied extracellularly. Cells pretreated with pertussis toxin (PtX) to selectively uncouple alpha i/alpha o type G proteins from their biological activators, however, did not respond to rab3 peptides. rab3-like peptides also induce a Ca2+ transient in mast cells. These observations provide evidence for functional coupling between an effector domain peptide sequence of rab3 protein and a PtX-sensitive G protein substrate.

Amino Acid Sequence↗
PubMed1991

Blot-sequencing of antibodies: application to analysis of V gene usage among anti-steroid monoclonal antibodies.

Automated gas-phase protein sequencing has been used to characterize variable regions of antibody heavy and light chains separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto Immobilon polyvinylidene difluoride membranes ('blot-sequencing'). Starting from 100 micrograms of antibody, 20 or more residues of N-terminal VH and VL sequences can regularly be obtained, which is often sufficient to assign the V region to a known family or subgroup. We have applied the blot-sequencing method to analysis of VH and VL usage among a panel of monoclonal anti-steroid antibodies, namely anti-progesterone, anti-pregnanediol, anti-estrone and anti-testosterone. The results demonstrate restricted, repetitive usage of VL subgroups and VH families related to anti-steroid specificities. VL regions of the VK1 group were particularly associated with anti-progesterone, VK21 with anti-estrone, and VK8 and VK9 with anti-pregnanediol. VH regions of anti-progesterone antibodies were all derived from the VHVGAM3.8 family; anti-estrone and anti-pregnanediol antibodies were derived from the VH7183 and VH36-60 families. The latter two families appear to characterize antibodies raised against steroids conjugated to proteins via a sugar bridge. Differences in VH/VL combination were associated with diversity of antibody specificity. In order to extend the sequence data obtained by this technique and confirm family assignments, we have shown that internal V-region sequences can be obtained by limited chemical cleavage of whole antibody with cyanogen bromide, followed by separation of individual fragments by SDS-PAGE and blot-sequencing.

Amino Acid Sequence↗
PubMed1990

Experimental hemiparkinsonism in the rat following chronic unilateral infusion of MPP+ into the nigrostriatal dopamine pathway--III. Reversal by embryonic nigral dopamine grafts.

In a previous study conducted over six months, we demonstrated that 1-methyl-4-phenylpyridinium ion (MPP+) the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, chronically infused (10 micrograms/24 h for seven days) into one median forebrain bundle of the rat can cause long-lasting damage to the nigrostriatal dopamine system. The present study was carried out in animals 18-19 months after MPP+ infusion to determine firstly, if the lesion was indeed permanent and secondly, if embryonic nigral dopamine suspension grafts implanted into the dopamine-denervated neostriatum can reverse the neurochemical and behavioural deficits induced by MPP+. All the animals within the MPP(+)-lesioned group showed robust contralateral and ipsilateral turning in response to apomorphine (0.05 mg/kg) and methamphetamine (2.5 mg/kg), respectively, at each time point of testing. In the grafted animals there was a progressive significant reduction in the number of rotations in response to both apomorphine and methamphetamine over the three-month test period. Autoradiographic analysis of [125I]sulpiride binding to striatal sections showed a 27% increase in dopamine D2 receptor density in the ipsilateral striatum of MPP(+)-lesioned animals. This increase in D2 receptor density was completely abolished by the dopamine grafts so that the D2 receptor density in the grafted striatum was similar to the contralateral striatum of MPP(+)-lesioned animals. This increase in D2 receptor density was completely abolished by the dopamine grafts so that the D2 receptor density in the grafted striatum was similar to the contralateral striatum of the grafted animals or the ipsilateral striatum of control non-lesioned animals. In all the animals of the lesioned and grafted groups there was a complete loss of dopamine neurons in the ipsilateral substantia nigra as demonstrated by tyrosine hydroxylase-immunohistochemistry and in-situ hybridization histochemistry. In all the animals that received nigral dopamine grafts, numerous cells were localized within the grafts which contained tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase mRNA. Moreover, immunohistochemical staining showed a dense network of tyrosine hydroxylase-positive fibres within the grafted striatum. The results of the present study are important in two respects. Firstly, they demonstrate that MPP+ infusions into the rat nigrostriatal dopamine pathway can produce a permanent degeneration of nigral dopamine neurons. Thus, in animals assessed 18-19 months after the initial MPP(+)-lesion there was no significant behavioural or neurochemical compensation with time. Secondly, the results clearly show that embryonic nigral dopamine grafts implanted into the dopamine-denervated striatum can reverse the behavioural and neurochemical deficits induced by MPP+.

1-Methyl-4-phenylpyridinium↗
PubMed1989

Automated enzymatic assays for the determination of intestinal permeability probes in urine. 2. Mannitol.

A need for a simple method for the determination of mannitol in urine has arisen because of the use of this monosaccharide in intestinal permeability tests. A rapid spectrophotometric assay for mannitol is presented based on the use of the bacterial enzyme mannitol dehydrogenase. The technique has been automated for use on the Cobas-Bio (Roche) centrifugal analyser. The assay has been shown to be highly specific for mannitol and was not affected by high concentrations of glucose, lactose or lactulose in samples. Within assay coefficient of variation was in the range 0.3 to 1.1% and 0.6 to 2.4% between assays. The technique represents a significant improvement in terms of time, simplicity and precision on existing methods.

Humans↗
PubMed1989

Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels.

A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-alpha family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

Amino Acid Sequence↗
PubMed1988

Insulin-like growth factor II in the rat choroid plexus.

In situ hybridization histochemistry has been used to demonstrate the expression of the mRNA for insulin-like growth factor II (IGF-II) in the adult rat choroid plexus. IGF-II mRNA was found in the choroid plexus cells by using two different probes specific for different parts of IGF-II sequence. Parallel studies on consecutive sections showed that message for a choroid plexus marker transthyretin mRNA was also localized in the same choroid plexus cells.

Animals↗
PubMed1987

Effect of insulin on hind-limb and whole-body leucine and protein metabolism in fed and fasted lambs.

1. A combination of isotope-dilution and arterio-venous difference techniques was used to determine rates of leucine metabolism and protein synthesis and degradation in a hind-limb preparation (predominantly muscle) and the whole body of eight lambs fed on milk to appetite and eight lambs fasted from 24 to 48 h. 2. Compared with fed lambs, fasted lambs showed decreased rates of protein synthesis in both whole body and hind-limb, and in hind-limb muscle, elevated rates of protein degradation. 3. The effects of two rates of insulin infusion on whole-body and hind-limb-muscle leucine metabolism, and in turn on protein metabolism, were determined. Insulin had no significant effect on leucine flux or oxidation (and hence protein synthesis and degradation) in whole-body or hind-limb muscle of fed lambs. In fasted lambs insulin progressively reduced arterial leucine concentration and whole-body leucine flux and oxidation, indicating a reduction in both protein synthesis and degradation. Insulin reduced the rate of leucine efflux from hind-limb muscle, which was followed by a reduction in leucine uptake. Insulin increased hind-limb-muscle glucose uptake in both fed and fasted lambs. 4. On the basis that hind-limb muscle was representative of skeletal muscle in general, we estimated that muscle accounted for the same percentage (about 27) of whole-body protein synthesis in both fed and fasted lambs. This percentage was unaffected by infusion of insulin, although the absolute rates differed in fed and fasted lambs.

Animals↗
PubMed1983

The metabolism of circulating non-esterified fatty acids by the whole animal, hind-limb muscle and uterus of pregnant ewes.

1. The over-all and regional metabolism of non-esterified fatty acids (NEFA) was studied using a combination of isotopic and arteriovenous-difference techniques. 2. There was a common linear relationship, whether stearic, palmitic or oleic acids were used as tracer, between the arterial NEFA concentration and the rates of entry and oxidation. 3. Assuming that the tracer used reflected the metabolism of all the NEFA, the total entry rate in fed and fasted pregnant ewes was (mean +/- SE) 0.44 +/- 0.02 and 0.55 +/- 0.07 mmol/h per kg body-weight respectively. Oxidation of NEFA contributed (mean +/- SE) 34 +/- 5 and 58 +/- 7% to the respiratory carbon dioxide in fed and fasted animals, this accounting for (mean +/- SE) 46 +/- 6 and 59 +/- 3% of the respective entry rates. 4. Hind-limb muscle both utilized and produced NEFA. The mean gross fractional extraction (calculated from isotopic uptake) was (mean +/- SE) 9 +/- 1%. Gross utilization of any NEFA and appearance of 14CO2 across the muscle were linearly related to the arterial concentration of tracer fatty acid, irrespective of whether this was oleate or stearate. The amount of 14CO2 appearing was consistent with (mean +/- SE) 54 +/- 8% of the CO2 produced by the hind-limb being derived from NEFA oxidation. 5. Infused NEFA were partly converted to ketone bodies. Uptake and oxidation in the hind-limb of ketones formed in the liver could account for approximately 20% of the 14CO2 apparently produced in muscle from NEFA. Correction for this reduces the proportion of CO2 derived from NEFA to 43%. There was some indication that ketones were also produced from NEFA in the hind-limb. 6. NEFA were not a significant energy source for the gravid uterus. 7. An over-all view of energy sources for the whole animal and for hind-limb muscle in normal and fasted pregnant sheep was presented.

Animals↗
PubMed1981

Acetate supply and utilization by the tissues of sheep in vivo.

1. The supply and utilisation of acetate has been estimated simultaneously in the whole animal and tissues of sheep using a combination is isotope-dilution and arteriovenous-difference techniques. Animals were made alloxani-diabetic and acetate metabolism was compared when stabilized to normal metabolite levels with insulin (ITA sheep) and when food and insulin had been withdrawn for 36 h (fasted, diabetic sheep). 2. Acetate was simultaneously produced and utilized by all tissues. The exogenous (or gut) supply of acetate was the most important determinant of circulating acetate level. Endogenous acetate was produced mainly in the liver; 77 and 94% in fasted, diabetic and ITA sheep respectively. The production of endogenous acetate remained fairly constant and was not related to ketogenesis, which supports the idea that circulating acetate is largely a produce of fermentation. The liver, gut and muscle utilized 17, 25 and 54% respectively (96% total) of the acetate entry rate in ITA sheep; a similar percentage utilization was found in fasted, diabetic sheep. 3. Acetate is largely oxidized to carbon dioxide in the gut and muscles of sheep and may account for 30-40% of their oxidative metabolism. This figure is similar to that for the whole animal. The total acetate taken up by the liver could account for 30% of the oxygen consumption; however, the liver may not directly oxidize all the utilized acetate. 4. The over-all conclusion from this study is that acetate is largely of dietary origin and the major factor determining its rate of utilization is the arterial concentration.

Acetates↗
PubMed1981

Diabolic acids: occurrence and identification in natural products and their metabolism by simple-stomached and ruminant animals.

1. A method for gas-liquid chromatographic analysis of the dimethyl esters of a homologous series of diabolic acids is described. 2. Only when combined with low-resolution mass spectrometry could the method be used to show unequivocally the existence of diabolic acids in natural products. 3. Diabolic acids occurred naturally in rumen and faecal lipids of all ruminants examined but could not be detected in a number of ruminant tissue samples. 4. Infused 14C-labelled (C16:0)2 diabolic acid was poorly absorbed by both simple-stomached and ruminant animals. Oxidation was more extensive in the ruminant, but there was no evidence of deposition of diabolic acid in the tissues of either animal.

Animals↗
PubMed1979

The low availability of dietary choline for the nutrition of the sheep.

1. Choline, which is present in the diet of the sheep either in the non-esterified form or combined in phospholipids, is rapidly degraded in the rumen. The ultimate product formed from the N-methyl groups is methane. 2. Analysis of the non-esterified choline and the phosphatidylcholine in ruminal and abomasal digesta indicate that the phospholipid is the main vehicle for the passage of choline to the lower digestive tract. 3. The concentration of phosphatidylcholine in abomasal digesta is lower than that of ruminal digesta, which is in line with a selective retention of protozoa in the rumen as observed by others. 4. On defaunation of the rumen to remove ciliated protozoa the concentration of phosphatidylcholine in ruminal digesta falls markedly and becomes lower than that in abomasal digesta. 5. Calculation shows that the adult sheep obtains at most only about 20--25 mg of effective choline per day from its diet (0.002--0.0025% of dietary total dry-weight intake). This is some fifty times less than the minimum required to avoid pathological lesions and death in other species investigated (0.1%+ of dietary dry-weight intake). 6. Sheep liver can synthesize choline from [14C]ethanolamine both in vitro and in vivo, but the synthesis of choline per kg body weight is many times less than it is in the rat. 7. The intact sheep oxidizes an injected dose of [1,2-14C]choline to CO2 at a rate that is several times less than that observed for the rat. This could help to explain the apparent minimal requirement of sheep for dietary choline.

Animal Nutritional Physiological Phenomena↗