RESUMO
BACKGROUND: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure-function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. RESULTS: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l(-1) from Escherichia coli. Chemical glycosylation with glycosyl-beta-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. CONCLUSIONS: Erythropoietin expressed in E. coli bearing specific Asn-->Cys mutations at natural glycosylation sites can be glycosylated using beta-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins.
Assuntos
Eritropoetina/metabolismo , Sequência de Aminoácidos , Eritropoetina/síntese química , Eritropoetina/genética , Escherichia coli/genética , Glicosilação , Humanos , Espectrometria de Massas , Oxirredução , Mapeamento de Peptídeos , Dobramento de Proteína , Proteínas RecombinantesRESUMO
Thirty patients with hypercalcaemia due to malignancy that persisted following rehydration, were treated with a single dose of the bisphosphonate, clodronate. Clodronate (1.5 g) was administered intravenously in 500 ml normal saline over 4 h. Serum and urine biochemistry were measured before and after treatment and the results were compared with data from 15 patients given the recommended regimen 300 mg intravenous clodronate daily for 5 consecutive days. The single infusion induced a rapid and significant fall in serum calcium, apparent at day 3 (P < 0.0001) that persisted to the end of follow-up at day 10 (P < 0.001). Eighty per cent (24/30) of patients became normocalcaemic. The response was associated with a significant decrease in fasting urinary calcium excretion, and no change in renal function, as judged by serum creatinine. The same dose of clodronate, given as 5 daily infusions, induced a comparable decrease in serum calcium, but was less rapid in onset so that at day 3 the serum calcium was significantly lower with the single infusion (P = 0.02). The calcium lowering effect of both regimens depended on the tumour type. We conclude that the single infusion of 1500 mg clodronate is as effective in reducing serum calcium as the same dose given over 5 days. The single infusion has a more rapid onset of effect, is more convenient than multiple infusions, and has no adverse effect on renal function.
Assuntos
Ácido Clodrônico/administração & dosagem , Hipercalcemia/tratamento farmacológico , Neoplasias/complicações , Adulto , Idoso , Cálcio/sangue , Esquema de Medicação , Feminino , Humanos , Hipercalcemia/sangue , Hipercalcemia/etiologia , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Neoplasias/sangueRESUMO
Bisphosphonates are widely used in disorders associated with increased resorption of bone, particularly in Paget's disease of bone and in the hypercalcemia of malignancy. Because of their undoubted efficacy and relatively low toxicity, bisphosphonates are attractive candidates for the management of osteoporosis. Clodronate, one of the many bisphosphonates being tested in osteoporosis, may be given intravenously or by mouth. In contrast to etidronate, even high doses of clodronate do not impair the mineralization of bone, making it suitable for long-term use in osteoporosis. As do all the bisphosphonates tested thus far, clodronate appears to delay the rate of bone loss in osteoporosis. Long-term studies are relatively few, so that its steady-state effects on bone mass are not yet known. Most data suggest clodronate is capable at least of delaying the rate of bone loss, but several pilot studies with this agent suggest that increments of bone mass might be sustainable for several years. Clodronate is likely to decrease the frequency of osteoporotic fractures, but there is no evidence for this at present. Well-controlled, long-term prospective studies are needed.
Assuntos
Remodelação Óssea/fisiologia , Reabsorção Óssea/tratamento farmacológico , Difosfonatos/uso terapêutico , Osteoporose/tratamento farmacológico , Ácido Clodrônico/uso terapêutico , Humanos , Fraturas da Coluna Vertebral/tratamento farmacológicoRESUMO
As laparoscopic cholecystectomy becomes more prevalent, the unexpected finding of common duct stones by operative cholangiography presents a therapeutic dilemma for the surgeon. We present our current techniques for laparoscopic cholangiography and management of choledocholithiasis, including the use of angioplasty balloons for ampullary dilation.