Different influences of two fractions of lung cytochrome b5 on reconstituted lung benzphetamine N-demethylase system.

Chromatography of lung microsomal cytochrome b5 obtained from DEAE-cellulose columns, yielded two distinct cytochrome b5 fractions. These cytochrome b5 fractions were further purified by Sephadex G-100 gel filtration chromatography. The specific cytochrome b5 content of fraction 1 and fraction 2 was found to be 16.5 and 16.4 nmol/mg protein respectively. Both fractions were free of cytochrome P-450, NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase activities. The effects of lung cytochrome b5 (fraction 1 and fraction 2) and liver cytochrome b5 on benzphetamine N-demethylase activity were examined. Four different reconstitution systems were used. Lung cytochrome b5 fraction 2 and liver cytochrome b5 stimulated N-demethylase activity in all four systems when b5:P-450 molar ratio was low, i.e. 0.25 or 0.5. Both cytochrome b5 samples inhibited N-demethylase activity when b5:P-450 ratio exceeded 1:1 molar ratio. In contrast lung cytochrome b5 fraction 1 stimulated N-demethylase activity in all four systems. Maximal enhancement of the activity was obtained when b5:P-450 ratio was 0.5. The greatest increase in N-demethylation activity was observed in the reconstitution system with the lowest concentration of cytochrome P-450 reductase, conditions which most closely resemble intact microsomes.

Functional outcomes after supracricoid laryngectomy.

OBJECTIVES: Local control and 5-year survival rates are similar for patients undergoing total laryngectomy and supracricoid laryngectomy for the treatment of advanced-stage laryngeal carcinoma. However, comprehensive studies of functional outcomes after supracricoid laryngectomy are lacking. STUDY DESIGN: Cohort study. METHODS: This investigation provides objective voice laboratory data, skilled listener impressions of voice samples, swallowing evaluations, and patient self-perceptions of speech ability obtained from 10 supracricoid laryngectomees. RESULTS: Results demonstrated variable acoustic and speech aerodynamic disturbances, hoarse-breathy vocal quality, and speech dysfluency. Patients' self-perceptions of voice revealed severe dysphonia that induced certain emotional, physical, and functional setbacks. However, blinded judges rated these individuals as possessing intelligible speech and communication skills. All patients demonstrated premature spillage of the bolus and varying degrees of laryngeal penetration, aspiration, and retention during swallowing studies. However, each patient used a compensatory strategy to protect the airway. Voice and swallowing abilities appeared to depend on the mobility of the arytenoid cartilages, base of tongue action, and residual supraglottic tissue for the creation of a competent neoglottal sphincter complex that vibrated during phonation efforts and protected the airway during deglutition. CONCLUSIONS: Supracricoid laryngectomy avoids the potential complications, limitations, and emotional problems associated with a permanent tracheostoma. All patients demonstrated intelligible voice and effective swallowing function postoperatively, supporting supracricoid laryngectomy as a suitable alternative surgical approach to the total laryngectomy in select patients.

Evaluation of hydroxyapatite ossicular chain prostheses.

Hydroxyapatite (HA) middle ear prostheses have gained popularity as an alternative to human autografts and homografts. This study reports on 3 HA prostheses types: total ossicular chain prostheses, used for grafting the stapes footplate to the tympanic membrane; partial ossicular chain prostheses, used for grafting the stapes superstructure to the tympanic membrane; and Kartush incus struts (Smith & Nephew Richards Inc), used for grafting the stapes superstructure to the undersurface of the malleus. This single-surgeon study of 33 consecutive cases revealed a statistically significant difference in mean postoperative air-bone gap and airbone gap closure between incus struts (14/26 dB) or partial (22/11 dB) or total (25/10 dB) ossicular chain prostheses (t test: P<0.05). Prognostic risk factors graded by the Middle Ear Risk Index indicate a tendency for worse postoperative hearing with increasing Middle Ear Risk Index. This study supports the use of HA ossicular prostheses and, in particular, the use of the malleus for ossicular chain construction.

Ontogenic development of corticotrophs in fetal buffalo (Bubalus bubalis) pituitary gland.

To evaluate the subpopulation of corticotrophs in developing buffalo (Bubalus bubalis) fetus, recovered pituitary glands (n=6 per group) from late first, second and third gestational female buffalo dams. The corticotrophs were identified by using specific antibodies against proopiomelanocortin (POMC) and adrenocorticotrophic hormone (ACTH) through immunohistochemistry. There was a significant (P≤0.05) increase of immunoreactive (ir) ir-ACTH cells during late 2nd trimester while, ir-POMC cells were more (P≤0.05) at late 3rd trimester of gestation as compared to other age groups. The quantity of co-localized cells for POMC and ACTH was significantly (P≤0.05) greater at the end of 1st gestation rather than 2nd and 3rd gestational fetal adenohypophyseal cells. This study is the first to demonstrate co-localization of POMC+ACTH and the affect of gestational age on the expression of these cells in buffalo fetus adenohypophysis.

Reduced expression of platelet surface glycoprotein receptor IIb/IIIa at hyperthermic temperatures.

BACKGROUND: Hyperthermic temperatures exist from the heat dissipation of the implantable energy source of an artificial heart. This procedure as well as therapies for cancer and thermal injuries pose a new medical problem. Among many reported effects of heat on biologic systems, platelet functions such as maximal aggregation and adhesion are known to be reduced. Using flow cytometry, we have studied platelet dysfunction at elevated temperatures and have gained a mechanistic comprehension of the loss of platelet function. EXPERIMENTAL DESIGN: Platelet rich plasma was incubated at differing temperatures for 1 hour. Immediately after, the platelets were stained using mAb against glycoprotein IIb/IIIa (GPIIb-IIIa) (CD41a) and other platelet surface glycoproteins (GP) involved in aggregation and adhesion. Relative fluorescence intensity was measured using single-labeled, laser flow cytometry to determine changes in GP surface expression. In addition, scanning electron microscopy was used to evaluate morphologic changes. RESULTS: Hyperthermic temperatures between 40 and 44 degrees C significantly lowered the mAb cell surface binding in vitro of GP that participate in aggregation and adhesion. The most dramatic temperature-dependent loss of mAb binding was demonstrated by anti-GPIIb-IIIa, the mAb against the fibrinogen receptor. mAb binding to this receptor at 44 degrees C was decreased to 6.2% of a base-line fluorescence intensity of 654 (arbitrary units). The ADP-induced aggregation of platelets incubated at the same temperature also decreased to 2.1% of maximum aggregation. Other mAb, such as those against the von Willebrand factor receptor (GPIb) (CD42b), the thrombospondin receptor (GPIV) (CD36), and GPIIIa (CD61), also showed statistically significant reduction of mAb binding but to a lesser degree. Finally, scanning electron microscopy as well as side-scatter density plots from flow cytometry revealed that platelets became more spherical after incubation at 44 degrees C. CONCLUSIONS: The significant reduction in mAb binding correlates with functional impairment exhibited during hyperthermic incubation. Our results support the loss of binding ability of surface GP that are involved in aggregation and adhesion as a mechanism of platelet dysfunction upon heating. GPIIb-IIIa appeared the most susceptible to heat and the principal agent in thermal induced loss of platelet function. Significant morphologic changes at 44 degrees C, the critical temperature at which ADP-induced aggregation ceases, may contribute as well.

Stimulatory effects of lung cytochrome b5 on benzphetamine N-demethylation in a reconstituted system containing lung cytochrome P450LgM2.

Cytochrome b5 was partially purified from sheep lung microsomes in the presence of detergents Emulgen 913 and cholate by three consecutive DEAE-cellulose and Sephadex G-100 gel filtration chromatographies. The specific content of cytochrome b5 was 16.5 nmol/mg protein and purified cytochrome b5 fractions were free of cytochrome P450, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase activities. The influences of increasing concentrations of lung cytochrome b5 on benzphetamine N-demethylation reactions were examined in four different reconstitution systems containing lung cytochrome P450LgM2, lung cytochrome P450 reductase and lipid. In each system concentration of reductase was doubled with respect to former system. In all systems cytochrome b5 stimulated benzphetamine N-demethylase activity especially when cytochrome b5 was present at 0.5:1 molar ratio with respect to cytochrome P450LgM2. Besides, the greatest fold of increase in benzphetamine N-demethylation activity due to addition of cytochrome b5 was observed in System 1 with the lowest concentration of reductase.

Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens.

Human monoclonal antibodies specific for the D antigen of the Rh system are valuable tools for blood group typing and prevention of erythroblastosis. In this study, peripheral blood lymphocytes obtained from an Rh-negative woman immunized with Rh-positive fetuses were immortalized with Epstein-Barr virus (EBV), and transformed lymphoblastoid cell lines (LCLs) secreting antibodies to Rh antigens were generated. The presence of specific antibody was assessed by direct haemagglutination using Rh-positive, papain-treated red blood cells (RBCs), and the production of human antibody was assayed by enzyme-linked immunosorbent assay (ELISA). Specificities of the antibodies were determined by a panel of RBCs of known Rh phenotypes. Five LCLs produced antibody specific for the D antigen, and one LCL showed specificity towards the C antigen of the Rh blood group system. High-titre anti-Rh antibody-producing LCLs were subsequently selected and fused with a human x mouse heteromyeloma cell line. A hybridoma line producing human antibody of the immunoglobulin M (IgM) isotype, which strongly reacted with the D antigen, was established. The hybridoma was cloned, and the monoclone has been stable for growth and antibody production during 8 months of continuous culture, with a mean antibody concentration of 11.5 microg mL-1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (Du); however, agglutination was achieved with papain-treated Du RBCs. Immunoprecipitation of the D antigen by this antibody, followed by Western blot analysis, did not reveal any immobilized D-specific polypeptide. As this human antibody readily agglutinates D+ RBCs in saline, it has the potential to be used as an efficient reagent in routine blood group typing.

The effect of endolymphatic sac excision in Menière disease.

The role of the endolymphatic sac in the pathoetiology of Menière's disease has been extensively studied but remains controversial. Likewise, the mechanism behind the efficacy of the endolymphatic-mastoid shunt procedure in Menière's disease remains obscure. The central hypothesis of this study is that the endolymphatic-mastoid shunt procedure is a destructive rather than a drainage procedure. Therefore removal of the extraosseous endolymphatic sac with scarring of the intraosseous portion may be more destructive and thereby more effective in the treatment of Menière's disease than conventional shunt placement alone in the extraosseous endolymphatic sac. This study reports a 2-year follow-up on the effect of complete removal of the extraosseous endolymphatic sac for 10 patients with intractable Menière's disease and 10 matched control Menière's disease patients undergoing conventional endolymphatic-mastoid shunt surgery. This study showed no statistical difference in the change in hearing, vertigo, or disability in the two groups.

Zones of atrial vulnerability. Relationships to basic cycle length.

BACKGROUND: Bradycardia is commonly found in individuals at risk for paroxysmal atrial fibrillation (AF). However, a clear relationship between lengthening of basic cyclic length (BCL) and AF has not been demonstrated. METHODS AND RESULTS: In 20 open-chest dogs, atrial refractoriness, AF vulnerability, and atrial activation times (ACTs) were determined in sinus rhythm and at BCLs of 400, 300, and 200 ms, and the findings at the same coupling intervals and stimulus strengths were compared. As BCL increased, AFV zone lengthened, and its outer limit occurred later in diastole. The outer limit of the AF vulnerability zone for a BCL was its relative refractory period; the inner limit, however, was not its effective refractory period. A border zone, defined by the inner limit of the AF vulnerability zone and the effective refractory period for a BCL, decreased as BCL lengthened, offsetting the increase in the AF vulnerability zone. The border zone was characterized by paradoxical stimulus current strength propagation relations and features suggesting supernormal conduction. ACT also increased with BCL lengthening. When AF induced by rapid atrial burst pacing was contrasted with AF induced by an extrastimulus, it tended to have a more disorganized pattern and lasted longer. CONCLUSIONS: Lengthening of BCL increases the AF vulnerability zone, extending its outer limit later in diastole and comprising an increasing component of the total duration of the relative refractory period. Very short BCLs create conditions that also favor AF vulnerability.

Frequency analysis of B lymphocytes specific for Rh antigens in naturally immunized Rh-negative women.

BACKGROUND AND OBJECTIVES: Despite a successful outcome of the anti-D prophylaxis programme, alloimmunization still occurs. The aim of this study was to estimate the frequency of Rh-specific B lymphocytes in the peripheral blood of nine Rh-alloimmunized individuals at different time intervals after parturition. MATERIALS AND METHODS: The donors' B lymphocytes were transformed with Epstein-Barr virus (EBV) and cultured at different cell densities over a feeder of human fetal fibroblasts. Culture supernatants were screened for human immunoglobulin by enzyme-linked immunosorbent assay (ELISA) and for anti-Rh antibody by using a direct haemagglutination technique. The percentage of CD19+ B lymphocytes in peripheral blood was determined by flow cytometry, and the frequency of Rh-specific B lymphocytes was estimated by limiting-dilution assay (LDA). RESULTS: The frequency of Rh-specific B lymphocytes varied from 1 : 150 to 1 : 27,850 in different donors. There was a decrease in this frequency and level of anti-Rh antibody with increase in time interval between bleeding and last exposure to the antigen. Furthermore, a positive correlation was observed between the titre of Rh-specific antibody and frequency of Rh-specific B cells in each of three subjects bled at multiple time-points postdelivery. CONCLUSIONS: The magnitude of the specific antibody response to Rh antigens varies greatly in Rh-alloimmunized women, which partly reflects the difference in frequency of specific B cells in these individuals.