Biomedical subjects
Publications and source records attributed to P Madsen.
A novel class of N,N-dibenzyl-N'-benzylidenehydrazines as potent and competitive glucose-6-phosphatase catalytic site inhibitors are described. Optimisation of this series identified compounds with IC(50) values as low as 170 nM.
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Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y.
We previously isolated and sequenced the approximately 250-kDa type 1 receptor sorLA/LR11, a mosaic protein with elements characterizing the Vps10p domain receptor family as well as the low density lipoprotein receptor family. The N terminus of the Vps10p domain comprises a consensus sequence for cleavage by furin ((50)RRKR(53)) that precedes a truncation found in sorLA isolated from human brain. Here we show that sorLA, like sortilin-1/neurotensin receptor-3, whose lumenal domain consists of a Vps10p domain only, is synthesized as a proreceptor that is cleaved by furin in late Golgi compartments. We show that the truncation conditions the Vps10p domain for propeptide inhibitable binding of neuropeptides and the receptor-associated protein. We further demonstrate that avid binding of the receptor-associated protein, apolipoprotein E, and lipoprotein lipase not inhibited by propeptide occurs to sites located in other lumenal domains. In transfected cells, about 10% of full-length sorLA were expressed on the cell surface capable of mediating endocytosis. However, the major pool of receptors was found in late Golgi compartments, suggesting possible interaction with newly synthesized ligands. The results show that sorLA, following activation by truncation, binds multiple ligands and may mediate both endocytosis and sorting.
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PURPOSE: Hypospadias incidence rates have been widely reported to be increasing. During the last 20 years there has been a significant increase in the number of women who delay childbearing until their mid 30s. Therefore, it was of interest to determine if increasing maternal age is an independent risk factor for hypospadias. MATERIALS AND METHODS: Data from the New York State Department of Health and California Birth Defects Monitoring Program were analyzed from 1983 to 1996 by maternal age groups of less than 20, 20 to 24, 25 to 29, 30 to 34, and 35 or greater years. A Poisson model was fitted to the data from each state using maternal age and year of birth from which relative rates were calculated. RESULTS: Our analysis revealed that advancing maternal age is significantly associated with hypospadias and is most evident for severe cases. For example, in California a 50% increase in severe cases was demonstrated for children of mothers older than 35 years compared to mothers younger than 20 years (p <0.05). CONCLUSIONS: Hypospadias is significantly associated with increasing maternal age. Women who elect to delay childbearing until their mid 30s or later should be aware that their offspring are at increased risk of hypospadias.
The discovery of the first class of potent glucose-6-phosphatase catalytic site inhibitors, substituted 4,5,6,7-tetrahydrothieno[3,2-c]- and -[2,3-c]pyridines, is described. Optimisation of this series involved solution phase combinatorial synthesis and very potent compounds were prepared with IC50 values down to 140 nM. The structure activity relationship (SAR) of these compounds indicates that: a tetrahydrothieno[3,2-c]pyridine core ring system and the isomeric [2,3-c] system are equipotent and much better than the corresponding benzo analogue, 1,2,3,4-tetrahydro-isoquinoline. The 4-substituent of the tetrahydrothieno[3,2-c]pyridine ring has to be a phenyl group, optionally substituted with a lipophilic 4-substituent, such as trifluoromethoxy or chloro. The 5-substituent of the tetrahydrothieno[3,2-c]pyridine ring has to be a substituted benzoyl; anisoyl and (E)-3-furan-3-ylacryloyl are the best of the investigated groups. Substitution in the benzoyl ortho position seems to be forbidden, whereas substitution in the meta position is tolerated only if a methoxy para substituent is present. These SAR findings were parallel to those obtained in the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine system. Enantioselectivity in enzyme recognition was observed and the activity resided in all cases only in one of the enantiomers.
Occasionally, lifting of a heavy weight leads to dizziness and even to fainting, suggesting that, especially in the standing position, expiratory straining compromises cerebral perfusion. In 10 subjects, the middle cerebral artery mean blood velocity (V(mean)) was evaluated during a Valsalva maneuver (mouth pressure 40 mmHg for 15 s) both in the supine and in the standing position. During standing, cardiac output decreased by 16 +/- 4 (SE) % (P < 0.05), and at the level of the brain mean arterial pressure (MAP) decreased from 89 +/- 2 to 78 +/- 3 mmHg (P < 0.05), as did V(mean) from 73 +/- 4 to 62 +/- 5 cm/s (P < 0.05). In both postures, the Valsalva maneuver increased central venous pressure by approximately 40 mmHg with a nadir in MAP and cardiac output that was most pronounced during standing (MAP: 65 +/- 6 vs. 87 +/- 3 mmHg; cardiac output: 37 +/- 3 vs. 57 +/- 4% of the resting value; P < 0.05). Also, V(mean) was lowest during the standing Valsalva maneuver (39 +/- 5 vs. 47 +/- 4 cm/s; P < 0.05). In healthy individuals, orthostasis induces an approximately 15% reduction in middle cerebral artery V(mean) that is exaggerated by a Valsalva maneuver performed with 40-mmHg mouth pressure to approximately 50% of supine rest.
The relationship between mastitis and functional longevity was assessed with survival analysis on data of Danish Black and White dairy cows. Different methods of including the effect of mastitis treatment on the culling decision by a farmer in the model were compared. The model in which mastitis treatment was assumed to have an effect on functional longevity until the end of the lactation had the highest likelihood, and the model in which mastitis treatment had an effect for only a short period had the lowest likelihood. A cow with mastitis had 1.69 times greater risk of being culled than did a healthy herdmate with all other effects being the same. A model without mastitis treatment was used to predict transmitting abilities of bulls for risk of being culled, based on longevity records of their daughters, and was expressed in terms of risk of being culled. The correlation between the risk of being culled and the national evaluations of the bulls for mastitis resistance was approximately -0.4, indicating that resistance against mastitis was genetically correlated with a lower risk of being culled and, thus, a longer functional length of productive life.
We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.
Central hypovolaemia induced by head-up tilt evokes a reduction in superior mesenteric artery resistance resulting in maintenance of regional blood flow. Mechanisms of importance for this response are not known, but a parasympathetic contribution could be expected. To evaluate this hypothesis, superior mesenteric artery blood flow and resistance were evaluated by duplex ultrasound in eight healthy volunteers during postprandial head-up tilt with and without cholinergic blockade. During supine rest, cholinergic blockade did not influence the postprandial reduction in peripheral mesenteric artery resistance as expressed by analogous elevations in the diastolic blood velocity (to 62 +/- 9 vs. 56 +/- 7 cm s-1 with placebo). Throughout the normotensive and hypotensive phases of head-up tilt, cholinergic blockade reduced mesenteric artery mean blood velocity by 39 and 42%, respectively, corresponding to volume flow reductions by 35 and 41% (0.62 +/- 0.10 vs. 0.96 +/- 0.13 L min-1 and 0.52 +/- 0.07 vs. 0.88 +/- 0.16 L min-1; P < 0.05). Also, during both phases of head-up tilt, cholinergic blockade increased mesenteric artery resistance as reflected in a reduction in the diastolic blood velocity by 41 and 56%, respectively (44 +/- 4 vs. 74 +/- 13 cm s-1 and 24 +/- 6 vs. 54 +/- 8 cm s-1). These results support a cholinergic contribution to the mesenteric artery vasorelaxing response to central hypovolaemia induced by head-up tilt.
Earlier studies of psoriatic and normal primary keratinocytes treated with phorbol 12-myristate-1-acetate identified two low-molecular-weight proteins, termed phorbolin-1 (20 kDa; pI 6.6) and phorbolin-2 (17.6 kDa; pI 6.5). As a first step towards elucidating the role of these proteins in psoriasis, we report here the molecular cloning and chromosomal mapping of phorbolin-1 and a related cDNA that codes for a protein exhibiting a similar amino acid sequence. The phorbolins were mapped to position 22q13 immediately centromeric to the c-sis proto-oncogene. Transient expression of the phorbolin-1 cDNA in COS cells and by in vitro transcription/translation, yielded polypeptides that comigrated with phorbolins-1 and -2. Comparative sequence analysis revealed 22% overall identity and a similarity of 44% of the phorbolins to apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme; however, recombinant-expressed phorbolin-1 exhibited no cytidine deaminase activity, using either a monomeric nucleoside or apolipoprotein B cRNA as substrate, and failed to bind an AU-rich RNA template. Whereas the precise function of the phorbolins remains to be elucidated, the current data suggest that it is unlikely to include a role in the post-transcriptional modification of RNA in a manner analogous to that described for apobec-1.
The combined effects of hyperventilation and arterial desaturation on cerebral oxygenation (ScO2) were determined using near-infrared spectroscopy. Eleven competitive oarsmen were evaluated during a 6-min maximal ergometer row. The study was randomized in a double-blind fashion with an inspired O2 fraction of 0.21 or 0.30 in a crossover design. During exercise with an inspired O2 fraction of 0.21, the arterial CO2 pressure (35 +/- 1 mmHg; mean +/- SE) and O2 pressure (77 +/- 2 mmHg) as well as the hemoglobin saturation (91.9 +/- 0.7%) were reduced (P < 0.05). ScO2 was reduced from 80 +/- 2 to 63 +/- 2% (P < 0.05), and the near-infrared spectroscopy-determined concentration changes in deoxy- (DeltaHb) and oxyhemoglobin (DeltaHbO2) of the vastus lateralis muscle increased 22 +/- 3 microM and decreased 14 +/- 3 microM, respectively (P < 0.05). Increasing the inspired O2 fraction to 0.30 did not affect ventilation (174 +/- 4 l/min), but arterial CO2 pressure (37 +/- 2 mmHg), O2 pressure (165 +/- 5 mmHg), and hemoglobin O2 saturation (99 +/- 0.1%) increased (P < 0. 05). ScO2 remained close to the resting level during exercise (79 +/- 2 vs. 81 +/- 2%), and although the muscle DeltaHb (18 +/- 2 microM) and DeltaHbO2 (-12 +/- 3 microM) were similar to those established without O2 supplementation, work capacity increased from 389 +/- 11 to 413 +/- 10 W (P < 0.05). These results indicate that an elevated inspiratory O2 fraction increases exercise performance related to maintained cerebral oxygenation rather than to an effect on the working muscles.
This review deals with glucagon receptor antagonism as a possible treatm ent of Type 2 diabetes. The role of glucagon in animal models has been studied by glucagon antibodies as model antagonists. Depending upon the animal model studied, selective glucagon deficiency produced by immunone utralisation suggests that glucagon plays a modest (rats) to substantial (rabbits) role in the maintenance of euglycaemia and is an important dia betogenic factor. These data strongly suggest that glucagon antagonism may be a beneficial and safe therapeutic approach for the treatment of T ype 2 diabetes. Further, the progress on non-peptide glucagon receptor antagonists is reviewed with special focus on the different classes of g lucagon receptor antagonists published, namely quinoxalines /pyrrolo[1,2 -a]quinoxalines, mercaptobenzimidazoles, 2-pyridyl-3,5-diarylpyrroles, q uinoline hydrazones, 4-phenylpyridines, and alkylidene hydrazides.
The first non-peptide competitive human glucagon receptor antagonist, 2-(benzimidazol-2-ylthio)-1-(3,4-dihydroxyphenyl)-1-ethan one, NNC 92-1687 (2), is described. This antagonist has a binding affinity of 20 microM (IC50) and a functional Ki = 9.1 microM at the human glucagon receptor. A structure-activity relationship (SAR) was obtained on this compound, and the results show that only the benzimidazole part can be changed without complete loss of affinity. Analogues with tert-butyl or benzyloxy groups in the 5-position of the benzimidazole moiety were found to be equipotent or slightly more potent, all displaying binding affinities around 5-20 microM. Most of the changes to the catechol and the linker gave compounds without any affinity toward the human glucagon receptor. The 3-hydroxy group could, however, in the presence of a 4-hydroxy group be changed to a methoxy or a chloro group while retaining affinity.
Knowledge about carbohydrate recognition domains of galectins, formerly known as S-type animal lectins, is important in understanding their role(s) in cell-cell interactions. Here we report the crystal structure of human galectin-7 (hGal-7), in free form and in the presence of galactose, galactosamine, lactose, and N-acetyl-lactosamine at high resolution. This is the first structure of a galectin determined in both free and carbohydrate-bound forms. The structure shows a fold similar to that of the prototype galectins -1 and -2, but has greater similarity to a related galectin molecule, Gal-10. Even though the carbohydrate-binding residues are conserved, there are significant changes in this pocket due to shortening of a loop structure. The monomeric hGal-7 molecule exists as a dimer in the crystals, but adopts a packing arrangement considerably different from that of Gal-1 and Gal-2, which has implications for carbohydrate recognition.
By microsequencing and cDNA cloning we have identified the transformation-sensitive protein No. IEF SSP 9302 as the human homologue of calumenin. The nucleotide sequence predicts a 315 amino acid protein with high identity to murine and rat calumenin. The deduced protein contains a 19 amino acid N-terminal signal sequence, 7 EF-hand domains and, at the C-terminus, a HDEF sequence which has been reported to function as retrieval signal to the ER. The calumenin transcript is ubiquitously expressed in human tissue, at high levels in heart, placenta and skeletal muscle, at lower levels in lung, kidney and pancreas and at very low levels in brain and liver. Calumenin belongs to a family of multiple EF-hand proteins that include the ER localized proteins reticulocalbin and ERC-55 and the Golgi localized Cab45. Since its Ca2+ binding may be important for the function of the protein we have used microdialysis experiments in order to analyse for the affinity and the capacity of recombinant human (rh) calumenin. All 7 EF-hands of the protein are functional and bind Ca2+, each with an affinity of 1.6x103 M-1. The relatively low affinity for the EF-hands may suggest a role for the protein in Ca2+-dependent processes in the ER.
Rab11a is a member of the rab-branch of the ras-like small GTP-binding protein superfamily that is associated with both constitutive and regulated secretory pathways. Using a direct procedure for cDNA cloning of small ras-related GTPases, that is based on the screening of eukaryotic cDNA expression libraries using [alpha-32P]GTP as a probe, we have isolated two cDNA clones encoding rab11a. Both clones share identical coding sequences, but differ in the length and sequence of their 3' untranslated regions (3'-UTR). Northern blot hybridisation analysis of various human tissues revealed indeed two mRNA species with lengths of 1.0 and 2.3 kb, respectively. Sequence analysis of the cDNAs identified two different putative polyadenylation signals (AATAAA) at positions 927 and 2302 of the larger transcript. In addition, the 3'-UTR of the larger transcript exhibited several AU-rich elements (ARE) that are believed to control gene expression by regulating the rate of mRNA degradation. Southern blots of human DNA digested with several rare restriction enzymes, and separated by pulse-field gel electrophoresis, yielded the same macro-restriction fragment pattern when hybridised with probes that discriminate between the two transcripts. Taken together, these findings imply that the two mRNA species originate from a single gene, which we have mapped to 15q21.3-q22.31, by the use of different polyadenylation sites. As expected, both rab11a-cDNAs yielded the same protein product when transiently expressed in COS-1 cells, and surprisingly, upregulated the proteome expression profile (de novo synthesis or posttranslational modification of preexisting proteins) of a few other, yet unknown GTP-binding proteins.