Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells

The native structures of the Asn-linked oligosaccharides and the O-glycans at Ser126 of human erythropoietin expressed from recombinant BHK cells have been elucidated. Enzymatically released N-glycans were studied by methylation analyses, fast-atom-bombardment mass spectrometry as well as one- and two-dimensional 1H-NMR spectrometry at 600 MHz. Many (82.7%) were found to be tetraantennary N-acetyllactosamine-type (22.8% with one, 3.6% with two and 0.4% with three N-acetyllactosamine repeats) being tetrasialylated (41%), trisialylated (29.6%) and disialylated (12.2%). A few (9.7%; 4.1% 2,4-branched, 5.6%, 2,6-branched) of the chains were triantennary (5.4% trisialyl, 4.3% disialyl) and 4.6% were of the disialyl diantennary type. Almost all of the innermost GlcNAc residues were alpha 1-6 fucosylated and NeuAc was exclusively alpha 2-3 linked to Gal beta 1-4GlcNAc-R; 60% of the protein was found to be O-glycosylated at Ser126; structures were monosialylated (70%) or disialylated (30%) forms of the Gal beta 1-3GalNAc core type. Glycosylation patterns at individual Asn-Xaa-Thr/Ser sites were determined by analytical high-pH anion-exchange chromatography with pulsed amperometric detection. Only tetraantennary chains with 0-3 N-acetyllactosamine repeats were detected at Asn38 and Asn83, while almost all of the di- and triantennary oligosaccharides were attached to Asn24. Batch analysis of different preparations of recombinant erythropoietin revealed the high reproducibility of the production procedure. Structures containing terminal GalNAc-GlcNAc were detected in small amounts in a few batches.     

Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization

The bacteriophage T7 DNA ligase gene was amplified using polymerase chain reaction-based methods and cloned into a T7 promoter-based expression vector. The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of protein per liter of bacterial culture. An initial physical and biochemical characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt-ended DNA fragments. Inhibition of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated. The enzyme has been crystallized from methoxypolyethylene glycol. The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 A. The unit cell dimensions are a = 66.1 A, b = 87.6 A, and c = 78.6 A, with one monomer in the asymmetric unit (Vm = 2.77 A3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized.     

Major depressive disorder: a prospective study of residual subthreshold depressive symptoms as predictor of rapid relapse

BACKGROUND: The study tested whether level of recovery from major depressive episodes (MDEs) predicts duration of recovery in unipolar major depressive disorder (MDD) patients. METHODS: MDD patients seeking treatment at five academic centers were followed naturalistically for 10 years or longer. Patients were divided on the basis of intake MDE recovery into residual depressive symptoms (SSD; N=82) and asymptomatic (N=155) recovery groups. They were compared on time to first episode relapse/recurrence, antidepressant medication, and comorbid mental disorders. Recovery level was also compared to prior history of recurrent MDEs ( > 4 lifetime episodes) as a predictor of relapse/recurrence. RESULTS: Residual SSD compared to asymptomatic recovery patients relapsed to their next MDE > 3 times faster (median=68 vs. 23 weeks) and to any depressive episode > 5 times faster (median=33 vs. 184 weeks). Residual SSD recovery status was significantly associated with early episode relapse (OR=3.65) and was stronger than history of recurrent MDEs (OR=1.64). Rapid relapse in the SSD group could not be attributed to higher comorbidity or lower antidepressant treatment. LIMITATIONS: Although inter-rater agreement on weekly depressive symptom ratings was very high (ICC > 0.88), some error may exist in assigning recovery levels. Antidepressant treatments were recorded, but were not controlled. CONCLUSIONS: MDE recovery is a powerful predictor of time to episode relapse/recurrence. Residual SSD recovery is associated with very rapid episode relapse which supports the idea that SSD is an active state of illness. Asymptomatic recovery is associated with prolonged delay in episode recurrence. These findings of this present study have important implications for the goals of treatment of MDD and for defining true MDE recovery.     

Validation of [13C]urea breath test for Helicobacter pylori using a simple gas chromatograph-mass selective detector

OBJECTIVE: Isotope ratio mass spectrometry (IRMS) is the accepted method for accurately measuring the 13CO2:12CO2 ratio in the non-invasive and non-radioactive [13C]urea breath test (13C-UBT) for Helicobactor pylori. The IRMS instrument, an expensive and highly specialized analyser, is rarely available. The objective of this project was to modify and validate the use of a simple bench-top gas chromatograph-mass selective detector (GC-MSD) for 13C-UBT. METHODS: Breath samples from 71 patients were taken at baseline and 30 min after ingestion of 100 mg [13C]urea. The breath samples were analysed using GC-MSD in the selected ion monitoring mode. The reference 13CO2:12CO2 ratio was from NBS19 obtained from the US National Institute of Standards and Technology. 13CO2:12CO2 ratios of the breath samples were determined. Excess delta per thousand (per mil, delta/thousand) of the 30 min sample over the baseline (deltadelta/thousand) of > or = 6deltadelta/thousand was considered H. pylori positive. Results from 13C-UBT and histology determined blind to each other were compared. RESULTS: The coefficient of variation of the reference 13CO2:12CO2 ratio was 0.06%. Using histology as the 'gold standard', the sensitivity (97.9%) and specificity (95.8%) of the GC-MSD 13C-UBT were comparable to those of other methods of H. pylori diagnosis. CONCLUSION: A gas chromatograph coupled to a mass selective detector that is available in many analytical and biomedical laboratories can be used for the 13C-UBT. This method will increase the availability and reduce the cost of this non-invasive, non-radioactive diagnostic test.     

Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the mouse

We analysed the occurrence of anti-neutrophil cytoplasmic antibodies (ANCA) in sera of 191 patients with glomerulonephritis (76 females and 115 males) by the standard indirect immunofluorescence method (IIF). The presence of ANCA was demonstrated in sera of 4.4% (8/181) patients with idiopathic glomerulonephritis (GN) and in 30% (3/10) of patients with rapidly progressive glomerulonephritis (RPGN), as a form of renal limited vasculitis. In the experimental part of our study we analysed the influence of GN ANCA-negative sera on the neutrophil function in vitro and compared with the effect of ANCA-positive sera (titre > or = 4:40) from systemic vasculitis (SV) patients with renal involvement. The activation of neutrophils was established by reactive oxygen species (ROS) production and the ability of superoxide anion to reduce ferrocytochrome c. Among 30 ANCA-negative GN sera 20% (6/30) revealed the ability to activate neutrophils isolated from healthy donor. Remaining ANCA-negative GN sera and all sera from normal healthy individuals (negative control group) did not affect the neutrophil function and did not induce the superoxide anion production. Their effect was similar to the second negative reference system without serum. Only 33% (3/9) of high titre ANCA-positive sera (> or = 1:40) from SV patients were able to activate neutrophils and to produce the superoxide anion with following ferrocytochrome c reduction, but the effect of activation was most powerfully expressed (three times greater than by GN ANCA-negative sera). The remaining ANCA-positive sera and all SV ANCA-negative sera did not affect the neutrophil function in vitro. These experimental data indicate that the presence of ANCA in GN sera is not necessary to induce neutrophil activation in vitro. On the other hand the influence of the SV ANCA-positive sera was most powerful expressed, although only 33% of sera were able to activate neutrophils in vitro. Our results indicate that not always ANCA presence in serum was connected with the ability to neutrophil activation in vitro. It is possible that in ANCA-negative sera other factors were able to activate neutrophils in vitro, but the effect of activation was markedly lower.     

Mature teratoma identified after postchemotherapy surgery in patients with disseminated nonseminomatous testicular germ cell tumors: a plea for an aggressive surgical approach

BACKGROUND: Mature teratoma is often found in resected retroperitoneal residual tumor masses (RRTM) after chemotherapy for disseminated nonseminomatous testicular germ cell tumors (NSTGCT). The aim of this report is to describe the clinical course of patients after resection of residual teratoma, with particular emphasis on relapse with either growing mature teratoma or secondary non-germ cell malignancy. METHODS: During the period 1979-1995, 113 patients underwent a laparotomy for resection of RRTM after chemotherapy for NSTGCT. Only patients with mature teratoma in the RRTM were included in the current study, and data on the patients who experienced relapse were studied in detail. RESULTS: Mature teratoma was found in 51 patients (45.1%) with RRTM resected after chemotherapy. Nine of these 51 patients (17.6%) relapsed; the relapses resulted from growing mature teratoma in 5 patients (9.8%), secondary non-germ cell malignancy in 3 patients (5.9%), and recurrent germ cell malignancy in 1 patient (2.0%). The primary treatment for all relapsing patients was surgical excision. All five patients with growing mature teratoma are alive without evidence of disease, as is the patient with recurrent germ cell malignancy. One of the three patients with non-germ cell malignancy died of disease, and the remaining two are alive with disease. CONCLUSIONS: Long term follow-up after resection of postchemotherapy residual teratoma is indicated because a proportion of patients develop growing mature teratoma or a secondary non-germ cell malignancy. The treatment for these recurrences should be complete surgical excision.     

Na+,K+-ATPase was found to be the membrane component responsible for the hydrophobic behavior of the brain membrane tubulin

We have previously described that the tubulin isolated from brain membranes as a hydrophobic compound by partitioning into Triton X-114 is a peripheral membrane protein [corrected]. The hydrophobic behavior of this tubulin is due to its interaction with membrane protein(s) and the interaction occurs principally with the acetylated tubulin isotype. In the present work we identified the membrane protein that interacts with tubulin as the Na+,K+-ATPase alpha subunit by amino acid sequencing. Using purified brain Na+,K+-ATPase we were able to isolate part of the total hydrophilic tubulin as a hydrophobic compound which contains a high proportion of the acetylated tubulin isotype.     

Endocrinological aspects of Langerhans cell histiocytosis complicated with diabetes insipidus

Langerhans cell histiocytosis (LCH) is a rare disorder and may be complicated with hypopituitarism and diabetes insipidus (DI) due to invasion of the hypothalamic-pituitary area. In this study, 10 patients with complete (4) and partial (6) type central DI were found among 125 LCH patients in our hospital records. The water deprivation test, followed by the pitressin test, was performed to confirm DI. Hypothalamic-pituitary endocrine function tests were carried out on these 10 patients at the initial diagnosis and during follow-up. All patients revealed growth hormone insufficiency in the insulin hypoglycemic tolerance test. Four patients had impairment of cortisol secretion, demonstrated by insulin hypoglycemic stimulating test results. Two patients had poor response in the thyrotropin releasing hormone stimulating test. Two patients had only partial responses in the luteinizing hormone releasing hormone test. Four patients had hyperprolactinemia. All patients underwent surgical treatment followed by chemotherapy and/or radiotherapy. One patient completely recovered from the endocrine disorder, 3 patients required smaller doses of desmopressin, and one patient had normal adrenal, thyroid, and gonadal function. Hypothalamic-pituitary disorders in LCH should not be neglected. Treatment of LCH can partially or completely reverse associated endocrine disorders. Therefore, endocrine studies and hormone replacement should be mandatory for patients with LCH.     

Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities

Npt2 encodes a renal-specific, brush-border membrane Na+-phosphate (Pi) cotransporter that is expressed in the proximal tubule where the bulk of filtered Pi is reabsorbed. Mice deficient in the Npt2 gene were generated by targeted mutagenesis to define the role of Npt2 in the overall maintenance of Pi homeostasis, determine its impact on skeletal development, and clarify its relationship to autosomal disorders of renal Pi reabsorption in humans. Homozygous mutants (Npt2(-/-)) exhibit increased urinary Pi excretion, hypophosphatemia, an appropriate elevation in the serum concentration of 1,25-dihydroxyvitamin D with attendant hypercalcemia, hypercalciuria and decreased serum parathyroid hormone levels, and increased serum alkaline phosphatase activity. These biochemical features are typical of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal Pi reabsorption. However, unlike HHRH patients, Npt2(-/-) mice do not have rickets or osteomalacia. At weaning, Npt2(-/-) mice have poorly developed trabecular bone and retarded secondary ossification, but, with increasing age, there is a dramatic reversal and eventual overcompensation of the skeletal phenotype. Our findings demonstrate that Npt2 is a major regulator of Pi homeostasis and necessary for normal skeletal development.