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.     

Distinction between artefactually shrunken and truly degenerated 'dark' neurons by in situ fixation with microwave irradiation

Dark, shrunken neurons frequently occur as artefacts in immersion fixed tissue. Perfusion fixation will prevent artefacts of this type. However, morphologically identical neurons have been described as truly degenerated cells in perfusion fixed brains in various pathological conditions. Since adequate perfusion is difficult to obtain in some pathological conditions, the question still remains whether the dark neurons found in some of these situations are true in vivo phenomena or artefacts caused by inadequate fixation. In the present study rat brains with cryogenic lesions were fixed in situ by microwave irradiation. With this method no artefactually changed dark neurons were observed in the normal parts of the brains. In the cryogenic lesions, however, a narrow rim of dark, shrunken neurons occurred adjacent to the normal cortex. This zone was identical to that observed in perfusion fixed tissue. Since inadequate fixation due to uneven perfusion of the damaged tissue is prevented with this method, we suggest that the neuronal changes represent true in vivo phenomena. Fixation with microwave irradiation can thus be used to differentiate between artefactually changed and truly degenerated dark neurons in various pathological conditions.     

Treatment of hemifacial spasm with botulinum toxin

We injected botulinum toxin to treat hemifacial spasm, and investigated the effects and the patient's impression of this treatment. Average duration of improvement lasted about 3.5 months in both the initial treatment group and the recurrent group. However the patients in the recurrent group received fewer units of botulinum toxin than those in the initial treatment group. Except for local paralysis that disappeared within a month, there were very few complications. Most patient were satisfied with this treatment. We conclude that the treatment of hemifacial spasm with botulinum toxin is both simple and useful.     

Asperger's syndrome--a separate nosologic entity or part of the spectrum of autism

This paper describes Asperger's syndrome, a disease similar to the autistic disorder, delineated for the first time by Asperger in 1944, just a few months after L. Kanner described infantile autism. Although, according to the epidemiological data, it occurs far more frequently than infantile autism, in practice this diagnosis is rarely established. Asperger's syndrome is defined as autistic syndrome among children with relatively high degree of intellectual functions, with marked disorder of speech and motoricity and interest confined to a very specified area. Learning is mechanical and routine and games uninventive. Children lack the need for age peer company. Boys are affected ten times more frequently than girls.     

Detection of Chlamydia trachomatis infections in women by Amplicor PCR: comparison of diagnostic performance with urine and cervical specimens

We used the Roche Amplicor PCR assay to compare urine and cervical swabs as sample material in the detection of Chlamydia trachomatis causing genital infections. The diagnostic performance of Amplicor PCR was compared with that of cell culture and the Gen-Probe PACE 2 assay with cervical specimens. If discrepant from other results, the specimens negative by PCR were diluted and reanalyzed to reveal PCR inhibitors. Of 666 patients, 39 (5.9%) were confirmed to have chlamydial infection. The respective sensitivity and specificity of Amplicor PCR were as follows: urine specimens, 82.0 and 99.7%; cervical specimens, 82.0 and 99.8%. Those for cell culture with cervical specimens were 84.6 and 100%. For the Gen-Probe PACE 2 assay, the sensitivity and specificity with cervical specimens were 79.5 and 100%, respectively. Without the effect of PCR inhibitors, the sensitivity of PCR with urine would have been 97.4%. Provided that the problems currently caused by inhibitors will be solved, the Amplicor PCR assay with urine specimens offers a tempting alternative for the diagnosis of C. trachomatis infection in women.     

Increased production of reactive oxygen species by rat liver mitochondria after chronic ethanol treatment

Rat liver microsomes and, to a lesser extent, nuclei were previously shown to produce reactive oxygen species at elevated rates after chronic ethanol treatment. The ability of intact rat liver mitochondria to interact with iron and either NADH or NADPH, and the effects of ethanol treatment, on production of reactive oxygen intermediates was determined. In the presence of ferric-ATP, NADH or NADPH catalyzed mitochondrial lipid peroxidation. Rates were elevated two- to threefold with mitochondria from ethanol-fed rats with both reductants. Mitochondrial lipid peroxidation was insensitive to superoxide dismutase, catalase, or hydroxyl radical scavengers but was sensitive to GSH and anti-oxidants such as trolox. Mitochondrial generation of hydroxyl radical-like species (assayed by oxidation of chemical scavengers) was increased after chronic ethanol treatment, as was H2O2 production. Modifiers of mitochondrial metabolism such as rotenone, cyanide, or an uncoupling agent, had no effect on mitochondrial production of reactive oxygen intermediates. The membrane-impermeable thiol reagent, p-chloromercuribenzoate, was complete inhibitory with both mitochondrial preparations. The activity of the rotenone-insensitive NADH-cytochrome c reductase, an enzyme of the outer mitochondrial membrane, was increased 40 to 60% by the ethanol treatment. These results suggest that NADH acting via the outer membrane NADH reductase can catalyze an iron-dependent production of oxygen radicals by rat liver mitochondria. The outer mitochondrial membrane fraction, prepared by digitonin fractionation, displayed increased rotenone-insensitive NADH-cytochrome c reductase activity after ethanol treatment and was more reactive in catalyzing scission of pBR322 DNA from the supercoiled form to the open circular forms. Rates of oxygen radical production by mitochondria and the extent of increase produced by chronic ethanol treatment are similar to those previously found with microsomes when NADH is the cofactor. Oxidation of ethanol by alcohol dehydrogenase generates NADH, and NADH-dependent production of reactive oxygen species by various organelles is increased after chronic ethanol treatment. These acute metabolic interactions coupled to induction by chronic ethanol treatment may play an important role in the development of a state of oxidative stress in the liver by ethanol.     

Experience with once daily dosing of gentamicin: considerations regarding dosing and monitoring

Plasma concentrations of gentamicin following a fixed dose of 240 mg once daily to patients with normal renal function were measured. The purpose was to establish guidelines to achieve a sufficiently high peak concentration with an appropriately low risk of accumulation. In 40 patients, 1-hour concentrations of plasma gentamicin had a median of 9.3 mg/l (range: 4.5-19.0 mg/l) and 9.7 mg/l (range: 3.6-14.6 mg/l) on days 1 and 3 of gentamicin treatment, respectively. Thirty-nine patients had 1-hour concentrations > 5 mg/l. The 1-hour concentrations varied considerably intra- and interindividually but showed a significant inverse correlation with body weight, surface area and the estimated endogenous creatinine clearance. The plasma gentamicin elimination half-life correlated significantly with age and inversely with body weight and creatinine clearance. There was no increase in the mean plasma creatinine from day 0 to day 4. No patients showed signs of nephrotoxicity, although 2 patients, both elderly and with low body weight, showed signs of beginning gentamicin accumulation. In conclusion, gentamicin treatment with the dose of 240 mg once daily in 3 days to adults with normal kidney function generally does not require adjustment or monitoring. However, the dose should be increased in young patients with an excessive body weight, and decreased doses are needed for old and underweight patients. Monitoring of trough plasma gentamicin concentration is not necessary with treatment duration of 3 days or less.     

Short-term preoperative radiotherapy results in down-staging of rectal cancer: a study of 1316 patients

OBJECTIVE: The incidence of back pain in children in our country is unknown. Several causes can produce this symptom, but it is necessary to rule out pathologies that require specific treatments. The goal of this work was to study the incidence and the etiology of back pain in children in our country. PATIENTS AND METHODS: We present a prospective study done in our Orthopedic Department during a period of 7 months. Twenty-two patients were referred with back pain of at least two months duration. The incidence was 2.9% compared to the total number of patients. The average age ranged from 7 to 17 years. RESULTS: A careful history and a radiological examination diagnosed 50% (11 cases) of the causes of backache in the present study. An idiopathic etiology was the most frequent cause and represented 9 patients (41%). In the remaining 12 cases (59%) pathological causes that required special treatment were identified including 2 cases of Scheuermann disease, 4 cases of painful scoliosis, 3 spondylolysis with spondylolisthesis, 2 cases of discitis, 1 benign neoplasm and 1 psychogenic cause. CONCLUSIONS: We recommend conservative management with the use of medication for relief of pain and physical therapy in the idiopathic cases. In the remaining cases, the specific treatment of the disease can improve the back pain.