Middle-Down Fragmentation for the Identification and Quantitation of Site-Specific Methionine Oxidation in an IgG1 Molecule

A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middle- down N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.     

ENDOSCOPY ASSISTED REMOVAL OF FERROMAGNETIC COINS WITH NOVEL MAGNETIC INSTRUMENT

Background: Ingestion of coins is a common clinical problem in children. Many of the coins are ferromagnetic and can be retrieved with the help of a magnet. We describe the use of a novel endoscopic accessory for removing ferromagnetic coins. Material and methods: Two magnet discs of 1.5 cm diameter were joined to a 200 cm steel wire of 0.75 mm thickness with a terminal 5 cm spring. A Teflon tube (160 cm, 7 F) was used along with this instrument as a sleeve. The use of this accessory was analyzed prospectively in subjects presenting with a history of coin ingestion. The time taken for removal of coins, complications during the procedure and failure rate was noted. Effect of the magnet on cardiac rhythm was also noted during the procedure. Results: A total of 55 children (mean age 5.1 ± 2.3 years) with coin ingestion presented over a period of 1 year. Forty‐four coins were ferromagnetic. All ferromagnetic coins were removed successfully. Mean time for removal was 68 ± 22 s. No complications were encountered. Conclusion: The novel magnetic instrument is precise, safe and quick for the removal of ferromagnetic coins under direct vision.     

Role and value of laparoscopic training devices in assessing nondominant and two-handed dexterity

Background and objectives: To determine improvement in hand dexterity with targeted laparoscopic skill exercises desirable for use in the operating rooms among in-training laparoscopic gynaecological surgeons and medical students. Design: Cross-sectional study with paired analysis. Setting: Kiel School of Gynaecological Endoscopy and Reproductive Medicine, Germany, between February and April 2005. Subjects: Twenty third-year medical students and 20 in-training gynaecological endoscopic surgeons from various parts of the world. Interventions: Demonstration and explanation of a set of five laparoscopic skill exercises desirable for use in the operating rooms before administering a pretest. This was followed by voluntary practice of these exercises for at least 10 times over 1 day. The posttest was performed the next day once the participant was comfortable performing the skill. Pre- and posttest assessments were conducted by independent supervisors. Main outcome measures: Time to completion of tasks with minimal errors. Results: There was significant reduction in mean time for all the laparoscopic skill exercises performed with dominant, nondominant, and both hands, before and after the training and practice (p-value <0.01; paired t-test). Moderate to high correlation (0.617–0.901) was seen with the intermediate and complex/difficult tasks, whereas low correlation was seen with the simple/easy task (0.200–0.336). Medical students and gynaecologists both showed improvement in performance from pretest to posttest in terms of reduction in mean time taken to perform all the tasks with minimal errors. Conclusions: Simple laparoscopic training devices can substantially help an individual hand’s improvement and acquisition of laparoscopic skills. Simple laparoscopic training devices along with animal models will continue to provide an efficient and effective environment for learning and teaching laparoscopic surgical skills. With this training, performance improves progressively with practice.     

Standardization of factor IX: standards for "purified" factor IX concentrates.

An international collaborative study was carried out to determine the suitability of the current WHO II-IX-X concentrate standard, 84/681, for assigning potency to the more highly purified factor IX concentrates. Three Coagulation Factor IX (Human) preparations and one Factor IX Complex preparation were assayed by the one stage method against WHO 84/681 following predilution to 1.0 u/ml in buffer, 1% albumin, or factor IX deficient plasma. There were no cases of non-parallelism between any of the preparations and the current WHO standard. Predilution of the Coagulation Factor IX (Human) preparations in 1% albumin or factor IX deficient plasma gave similar potency values. Predilution in buffer gave significantly lower (p less than 0.01) potency values. For the Factor IX Complex preparation, potency estimates were significantly different (p less than 0.01) with each prediluent. The overall precision was similar within each predilution for all preparations with predilution in buffer being less precise than predilution in albumin or in deficient plasma. WHO standard 84/681 appears to be a suitable standard for the potency determination of the more highly purified factor IX preparations. Predilution in 1% albumin or factor IX deficient plasma is recommended as they give equivalent results with the least variability.     

Clinical profile of trichotillomania.

Twenty-four cases of trichotillomania attending psychiatry outpatient department and child guidance clinic at Kalawati Saran Children's and Smt Sucheta Kriplani Hospitals over a period of 2 years from July, 1985 to November 1987 were studied. Females (66.7%) outnumbered the males (33.3%). Majority of cases belonged to age group 6-10 years (54.2%) and nuclear family (68.5%). Nail-biting (25.0%) was the commonest associated neurotic trait, followed by enuresis (20.9%), temper-tantrum (12.5%), etc. A past history of hysterical fits and neurotic depression was found in 3 cases (12.5%) and 2 cases (8.3%) respectively. Family history of neurosis was seen in mothers and fathers of 20.9% and 12.5% cases respectively. Trichobezoars and trichophytobezoars were found in 6 cases (25.0%) and 3 cases (12.5%) respectively. Majority of patients of trichobezoars presented with vague complaints like heaviness in the stomach (55.6%), inability to gain weight (44.4%), etc, while 22.2% cases were asymptomatic and detected only on screening.     

Anticonvulsant profile of drugs which facilitate GABAergic transmission on convulsions mediated by a GABAergic mechanism

The effects of selected modulators of GABAergic transmission, either alone or in combination, were tested for their potency on the seizure pattern and mortality induced by convulsant drugs in rat. Pentobarbital and diazepam were effective against both tonic and clonic seizure components induced by bicuculline and picrotoxin. The anticonvulsant profile of ethanol closely resembled that of pentobarbital. Pentobarbital, diazepam and ethanol did not modify seizures induced by strychnine. In contrast, progabide, a central gamma-aminobutyric acid (GABA) receptor agonist, caused significant protection only against convulsions induced by strychnine and its lethality, but did not protect against seizures induced by bicuculline or picrotoxin. Data on interaction of drugs with subprotective doses of these agents clearly demonstrated potentiation of the anticonvulsant actions of these modulators. Thus, seizures induced by bicuculline were more sensitive to the inhibition by pentobarbital in combination with diazepam or ethanol, while pentobarbital with progabide was equally effective against convulsions induced by GABA antagonists. Diazepam, in combination with progabide, blocked only convulsions induced by picrotoxin. Ethanol, in combination with either pentobarbital or with diazepam, was effective against all the three convulsant drugs. These results are consistent with the concept that drugs which facilitate GABAergic transmission are effective against seizures related to an impairment of GABA transmission. Further, the present data indicate that tonic seizures are more susceptible to the actions of drugs than the clonic component. Smaller doses of these drugs, alone or in combination, modified the seizure patterns and mortality, whereas at larger doses, the possible involvement of a nonspecific depressant action cannot be ruled out.     

Psychological responses to thermal stress in a glass bangle factory.

Behavioural studies were conducted on a random sample of 38 glass bangle workers engaged in various occupations and exposed to high ambient temperature (38.2 +/- 3.4 degrees C) and radiant heat (46.2 +/- 5.1 degrees C) during their work in the glass bangle industries at Firozabad, India. Twenty-two control subjects of similar age and socio-economic status unexposed to heat during their work were also studied for comparison. The digit span test, digit symbol test, Bourdon-Wiersma vigilance test, flicker fusion test and Maudsley personality inventory (MPI) were used to assess the behavioural changes induced by the existing heat stress. The mean scores of immediate memory were much lower in the exposed population than in the controls thereby indicating impairment of immediate memory in the study group. The visuo-motor coordination and vigilance were also significantly affected. Visual perception showed a downward trend among the glass bangle workers, although the MPI test indicated no personality changes.     

Potential drug targets for Mycobacterium avium defined by radiometric drug-inhibitor combination techniques.

Previously established radiometric techniques were used to assess the effectiveness of combined antimicrobial drug-inhibitory drug (drug-inhibitor) treatment on two clinical isolates of the Mycobacterium avium complex representing three colony variants: smooth opaque (dome) (SmO), smooth transparent (SmT), and rough (Rg). All variants were identified as members of the M. avium complex; however, only the SmT colony type of strain 373 possessed characteristic serovar-specific glycopeptidolipid (GPL) antigens. MICs, determined radiometrically, of drugs with the potential to inhibit the biosynthesis of GPL antigens or other cell envelope constituents were similar for all strains. These drugs included cerulenin, N-carbamyl-DL-phenylalanine, N-carbamyl-L-isoleucine, trans-cinnamic acid, ethambutol, 1-fluoro-1-deoxy-beta-D-glucose, 2-deoxy-D-glucose, and m-fluoro-phenylalanine. The MICs of the antimicrobial drugs amikacin, sparfloxacin, and clarithromycin varied, but overall the MICs for the SmO variant were the lowest. Radiometric assessment of drug-inhibitor combinations by using established x/y determinations revealed enhanced activity when either ethambutol or cerulenin were used in combination with all antimicrobial agents for all variants except the Rg variant of strain 424, for which ethambutol was not effective. Enhanced activity with amino acid analogs was observed with the Rg colony variants of strains 373 and 424. Two potential sites for drug targeting were identified: fatty acid synthesis, for all strains assayed, and peptide biosynthesis, particularly for Rg colony variants that possess previously identified phenylalanine-containing lipopeptides as potential targets for future drug development.