Production of recombinant human erythropoietin from Pichia pastoris and its structural analysis.

AIMS: To design and investigate a recombinant expression system producing a therapeutically important glycoprotein, human erythropoietin (rHuEPO), by Pichia pastoris. METHODS AND RESULTS: EPO cDNA was cloned into pPICZalphaA for expression under control of AOX1 promoter and fused, on the amino-terminal end, with a polyhistidine tag for rapid purification. A target site for factor Xa protease was also introduced, such that cleavage in vitro produced a mature form of rHuEPO having the native N- and C-termini. RHuEPO was characterized as to the extent and nature of N-linked glycosylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and western blotting. The rHuEPO produced was approximately 30 kDa. All three N-linked glycosylation sites were occupied dominantly by Man(17)(GlcNAc)(2). N-glycanase-treated rHuEPO purified but not digested with factor-Xa-protease, showed a spectral peak centered about m/z 20400 Da. CONCLUSIONS: The native polypeptide form of human EPO (c. 18 kDa) was obtained for the first time in P. pastoris expression system, after affinity purification, deglycosylation and factor-Xa-protease digestion. The amount of sodium dodecyl sulfate used prior to deglycosylation was found to be crucial in determining the dominant form of glycan in glycoproteins. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel approaches to protein expression and purification system and structural analysis presented, would be important especially for therapeutic proteins expressed in P. pastoris.

Hearing loss due to mannosidosis and otitis media with effusion. A case report and review of audiological assessments in children with otitis media with effusion.

A case of a child with mannosidosis and bilateral otitis media with effusion (OME) is reported here along with some discussion of relevant literature to emphasize the need for age appropriate audiometric assessment before and after insertion of grommets for glue ear (OME). There is a need for multidisciplinary teamwork in the management of children with hearing loss. If OME is treated surgically, age-appropriate hearing assessment is required before and after insertion of grommets. The need for audiological assessments will be relevant even if children had passed the newborn hearing screening test.

Microtubule-associated protein kinase-2 phosphorylates and activates tyrosine hydroxylase following depolarization of bovine adrenal chromaffin cells.

Depolarization of cultured bovine adrenal chromaffin cells with KCl increased the activity of a proline-directed protein kinase that phosphorylates tyrosine hydroxylase. Characterization of the KCl-activated protein kinase activity revealed that it shared similar biochemical and chromatographic properties with the microtubule-associated protein-2 kinase/extracellularly regulated kinase (MAP/ERK) family of protein kinases. This protein kinase activity was found to elute from Mono Q, Superose, and phenyl-Sepharose columns under conditions described for MAP/ERK kinases, and active fractions were found to react with specific antibodies directed against ERKs. The KCl-activated protein kinase was found to phosphorylate the serine 31 site of endogenous bovine adrenal tyrosine hydroxylase. This phosphorylation resulted in an approximately 2-fold activation of tyrosine hydroxylase.

Proline-directed phosphorylation of human Tau protein.

The primary sequence of the microtubule-associated protein tau contains multiple repeats of the sequence -X-Ser/Thr-Pro-X-, the consensus sequence for the proline-directed protein kinase (p34cdc2/p58cyclin A). When phosphorylated by proline-directed protein kinase in vitro, tau was found to incorporate up to 4.4 mol of phosphate/mol of protein. Isoelectric focusing of the tryptic phosphopeptides demonstrated the presence of five distinct peptides with pI values of approximately 6.9, 6.5, 5.6-5.9, 4.7, and 3.6. Mapping of the tryptic phosphopeptides by high performance liquid chromatography techniques demonstrated three distinct peaks. Data from gas phase sequencing, amino acid analysis, and phosphoamino acid analysis suggest that proline-directed protein kinase phosphorylates tau at four sites. Each site demonstrates the presence of a proline residue on the carboxyl-terminal side of the phosphorylated residue. Two phosphorylation sites are located adjacent to the three-repeat microtubule-binding domain that has been found to be required for the in vivo co-localization of tau protein to microtubules. Two other putative phosphorylation sites are located within the identified epitope of the monoclonal antibody Tau-1. Phosphorylation of these sites altered the immunoreactivity of tau to Tau-1 antibody. Since the neuronal microtubule-associated protein tau is multiply phosphorylated in Alzheimer's disease, and Tau-1 immunoreactivity is similarly reduced in neurofibrillary tangles and enhanced after dephosphorylation, phosphorylation at one or more of these sites may correlate with abnormally phosphorylated sites in tau protein in Alzheimer's disease.

Branhamella catarrhalis colonization in preschool asthmatics.

Branhamella catarrhalis has been associated with exacerbations of chronic bronchitis and asthma in adults. To investigate the possible role of B. catarrhalis in asthma of early childhood, we took posterior pharyngeal swabs from 24 normal children, 20 well asthmatics, and 20 acutely wheezy asthmatics, all between 1 and 4 years of age. On culture, 33% of the normal children were colonized with B. catarrhalis; colonization rates in the well asthmatics (70%) and in the wheezy asthmatics (75%) were significantly higher than in normals. The nature of this association requires further study.

Accident and emergency department attendance by asthmatic children.

Attendances at the accident and emergency department of a children's hospital for treatment of acute asthma were studied for one year to determine the characteristics of the children attending and their management. Eight hundred and twenty children, median age 5.5 years, made 1389 visits. Records were available from 1046 visits. Clinical information and assessment of the severity of the attack in the department was often inadequate. Peak flow records were available for 366 (35%). Attendances were most frequent in September and during the evening, but there was no significant day to day variation. Eight hundred and three children (78%) were self referred. Before attendance 962 (92%) had used a bronchodilator, including nebulised salbutamol (11%); 2% had taken prednisolone and 21% antibiotics. Five hundred and sixteen visits (49%) led to admission and 19% of those admitted required intravenous treatment. Probably some children who at present attend hospital for treatment of acute asthma could be managed at home, but this cannot be assumed without better understanding of the reasons for hospital attendance. More information is needed.

Recurrent accident and emergency department attendance for acute asthma in children.

Asthmatic children aged over 5 years making repeated visits to the accident and emergency department of a children's hospital were compared prospectively, on the basis of a clinical questionnaire and pulmonary function tests, with a control group of outpatients with asthma to find the reasons for their repeated attendance. Recurrent attenders (n = 145) had more severe asthma than control subjects (n = 118), with greater airway obstruction at rest (FEV1 79% v 85% predicted) and bronchial lability (47% v 38%). Significantly more of the "emergency" group used pressurised aerosols and fewer dry powder inhalers to administer bronchodilators. There were no differences in prophylactic treatment. Seventy one per cent of parents in the emergency group had feared that their child would die during an attack, compared with 56% of control subjects. Eighty one per cent of children were self referred to the accident and emergency department. Most parents had found hospital to be the quickest means of obtaining treatment in an emergency. There were no differences between the two groups in parents' knowledge about asthma, home conditions, or social disadvantage. Although children who repeatedly attend hospital accident and emergency departments for treatment of acute attacks have more severe asthma than controls and show some deficiencies in treatment, the major determinant of attendance appeared to be the parents' conviction that appropriate treatment could not be obtained elsewhere.

HLA type, islet cell antibodies, and glucose intolerance in cystic fibrosis.

Impaired glucose tolerance, assessed by a raised glycated haemoglobin (HbA1) concentration, was found in 24 (39%) out of 61 patients with cystic fibrosis with an age range of 1-23 years. No correlation between age and HbA1 concentration was found indicating that factors other than progressive pancreatic fibrosis may be important in the aetiology. HLA typing, islet cell antibodies, and autoantibody screen were completed. Eighteen (75%) out of 24 patients with cystic fibrosis who had an impaired glucose tolerance had HLA-DR3 or HLA-DR4 antigens compared with 23 (62%) out of 37 patients with normal glucose tolerance. Islet cell antibodies were present in seven (15%) out of 46 patients with cystic fibrosis; the prevalence in a normal population is 0.5%. Five (25%) of the 20 patients with a raised HbA1 concentration were positive for islet cell antibodies compared with two (8%) out of the 26 with normal glucose tolerance. Six (86%) out of seven patients who were positive for islet cell antibodies had HLA-DR3 or HLA-DR4 antigens. There was no general autoantibody production. Islet cell antibodies may play a part in the development of glucose intolerance in some patients with cystic fibrosis by being produced in those who are genetically predisposed as part of an immune response to damaged pancreatic tissue.

Characterization of recombinant human epidermal growth factor produced in yeast.

Four different forms of human epidermal growth factor (h-EGF) are found in the culture medium of a recombinant strain of Saccharomyces cerevisiae. These forms were characterized after purification using reverse-phase high-performance liquid chromatography. The most abundant form of secreted recombinant h-EGF has leucine at the carboxyl terminus and is identical with gamma-urogastrone. A second species is identical with the most abundant form except that it lacks the carboxyl-terminal leucine. This form appears to be the product of a carboxypeptidase found in the growth medium. The other two forms of recombinant h-EGF are the respective oxidation products of the above where the single methionine residue has been converted to methionine sulfoxide. These four forms of recombinant h-EGF are fully active; they bind to the EGF receptor of A431 cells as well as stimulate mitotic activity of human foreskin fibroblasts with equal specific activity. The location of the disulfide bonds in the predominant form of recombinant h-EGF was determined following digestion with thermolysin. The amino acid compositions of the resulting peptides showed that the placement of disulfide bonds in recombinant h-EGF is identical with that in murine EGF.

Gastrografin in acute meconium ileus equivalent.

Twenty-five (37%) patients with cystic fibrosis attending our clinic have experienced acute meconium ileus equivalent. In one year 37 of 40 episodes were treated with single dose oral Gastrografin with an 81% success rate, 75% being treated as outpatients. Patients found this treatment preferable to other recommended treatment.