Contours and distribution of sites that react with antiacetylcholinesterase in chicken intrafusal fibers

Serial cross and longitudinal sections of intrafusal fibers from the intracapsular portions of chicken tibialis anterior muscle spindles were incubated with a monoclonal antibody specific for chicken acetylcholinesterase (AchE) and examined by immunofluorescence for the presence of the enzyme on presynaptic and postsynaptic membranes of neuromuscular junctions. The midequatorial sensory region which lacks organized sarcomeres was negative, but immediately distal to it faintly staining regions of AchE localization were observed on intrafusal fibers. In cross sections at the juxtaequator, the outlines of areas that were positive for AchE were either thin and crescentlike or thick and compact. The distribution of both types of localization continued into the polar region. Toward the more distal polar region, the intensity of sites on the postsynaptic membrane that reacted with the anti-AchE progressively increased. In longitudinal sections, AchE localization was largely limited to two configurations. One was elongate, while the other was more round or oval and often also smaller. Both types might occur on the same, or on different, intrafusal fibers. Examination of silver-impregnated sections revealed the presence of platelike and of traillike axon terminals. The variety of shapes observed on presynaptic and postsynaptic membranes warrants further study to determine whether chicken muscle spindles are innervated by more than one type of motor neuron.     

Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice.

Monoclonal antibodies were raised against a bacterial fusion protein containing amino acids 364 to 623 of the major nonstructural protein, NS-1, of minute virus of mice (MVMp), an autonomous parvovirus. By immunoblot analyses, these antibodies all recognized an 83-kDa protein in MVM-infected mouse fibroblast cells. Indirect immunofluorescence studies showed that five of the six react against a nuclear protein in MVM-infected mouse cells resulting in discrete foci of fluorescence. These foci do not correspond with the nucleoli, the site of MVM DNA replication. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen and showed that four distinct epitopes were recognized by the different antibodies. A 25-amino-acid peptide was used in competition ELISAs to confirm the location of the epitope recognized by two antibodies CE10 and AC6. Preliminary characterization of an NS-1/NS-2 fusion protein synthesized in insect cells using a baculovirus expression vector showed that this fusion protein is also localized within the nucleus; however, in contrast, the full-length NS-1 polypeptide is located within the cytoplasm.     

Immunoassays and nucleic acid detection with a biosensor based on surface plasmon resonance

A technique based on surface plasmon resonance is described which can be used to detect changes of refractive index that occur when one partner of a molecular binding pair diffuses from solution to bind the other partner which is immobilised on a silver surface. Results for the molecular binding pairs; protein-antibody, hapten-antibody and DNA-DNA are described. Instrumentation necessary for implementation of the technique is detailed. Immunoassay of proteins and haptens is possible in less than one minute with a sensitivity of 10(-9) mol/l. Hybridisation of 10 fmoles of a 97 base target sequence on the 1 mm2 area of detection to an immobilised oligonucleotide probe can be detected in less than five minutes. Advantages of the technique include the ability to record the kinetics of binding reactions in "real time" and the lack of labels in this simple assay format. Methods of improving the sensitivity are discussed.     

Die pH-Aktivitätsspektren der sauren proteolytischen Enzymaktivitäten von Harn und Serum

Zusammenfassung 1. Die p_H-Abhängigkeit der sauren proteolytischen Enzymaktivitäten von Harn und Serum wurde mit Hämoglobin und Casein als Substraten mittels zweier unabhängiger analytischer Methoden im Bereich von p_H 0,9–4,4 untersucht. Die p_H-Aktivitätskurven von Harn und Serum wiesen hinsichtlich der Form, der Lage und der Ausprägung von Aktivitäts-Maxima deutliche Unterschiede auf. Die mittlere Aktivität der Seren betrug etwa ein Drittel der mittleren proteolytischen Aktivität der Harne.2. Die saure proteolytische Enzymaktivität des Harnes wurde durch Zusatz einer Heparin-Präparation gehemmt. Das Maximum der Hemmung lag zwischen p_H 2,2 und 3,0.3. Die saure proteolytische Enzymaktivität des Harnes zeigte sich bei p_h 3,50 alkalistabiler als diejenige bei p_H 2,15.4. Die Befunde werden im Hinblick auf die Theorie einer exogen-endogenen Partition der Enzyme des Gastrointestinaltraktes und die Heterogenität der sauren gastrointestinalen Proteasen diskutiert. Es wird geschlossen, daß die Bestimmung einer renalen Protease-Clearance wegen fehlender Zuordnungsmöglichkeit der proteolytischen Enzymaktivitäten von Harn und Serum zu bestimmten Enzymen bzw. Proenzymen noch nicht möglich ist.     

Bounds in the Sensitivity of BioMEMS Devices for Cell Detection

This paper presents an ongoing effort to characterize performance and reliability of micro electromechanical systems used for biomedical diagnostics (BioMEMS). In order to study the interactions of human osteosarcoma (HOS) cells with BioMEMS devices, cultures were performed on silicon (Si) surfaces as well as silicon surfaces coated with 50 nm of titanium (Ti). Cell spreading on the surfaces was observed over time for up to 2 hours. It was seen that titanium coated silicon surfaces have the potential to provide a better interface for BioMEMS devices, due to enhanced adherence and spreading of the cells on these surfaces. Atomic force microscope (AFM) cantilevers were used as cell detection sensors. These cantilevers were coated with 50nm of titanium metal to provide a cell friendly surface. Theoretical models were then developed for the prediction of the vibrational responses of the AFM cantilevers before and after cell attachment. The models were used to relate the experimentally observed changes in frequency to the number of cells that are attached on the cantilever. The bounds in the possible frequency changes were determined within a theoretical framework. From experimentally calculated values for the mass of cells, random number simulations were carried out to determine the probability of cell attachment as a function of the change in resonance frequency of the cantilever sensor. The implications of the results are then discussed for the future reliability modeling of the sensor.     

Characterization of non-homogeneous polymers with size-exclusion chromatography by implementing an on-line viscometer

The viscosity distribution of a polymer sample can be obtained by using an on-line viscometer as a detector in size-exclusion chromatography. This newly defined viscosity distribution is closely related to the molecular weight distribution and expresses weight fraction times intrinsic viscosity of species i as a function of the corresponding molecular weight times intrinsic viscosity (w_i[η_i] vs. M_i[η_i]). The intrinsic viscosity ([η]) and number-average molecular weight (M?_n) can be obtained directly from a viscosity distribution. If the Mark-Houwink exponent a is known (or approximately known) for non-homogeneous polymer the M?_w/M?_n can be estimated from the viscosity distribution when the molecular weight distribution is approximated with a known distribution function. These estimates are independent of any other detector and are valid even for non-homogeneous polymer samples. The relation between the moments of the viscosity distribution and the M?_w/M?_n is presented for two widely used distribution functions, the Log-Normal and the Generalized Exponential Distributions. Polymer characterization based on the viscosity distribution is shown to be a robust technique. It is particularly attractive in characterizing non-homogeneous polymers since it is solely obtained from on-line viscometer.     

A pilot study of recombinant interferon beta (IFN-βser) in patients with recurrent glioma

Recombinant interferon beta (IFN-_ser) has been administered by intravenous bolus injection three times weekly at a dose of 90 × 10⁶ IU to 14, patients with recurrent malignant glioma in an ongoing study. The treatment period has ranged from 1 to 40 weeks. The most common adverse experiences were fever, chills, malaise, and headache. Fever, chills and headache were worse with the first two doses and were usually relieved with acetaminophen. All patients tolerated subsequent treatments without any difficulties. No neurologic or hematologic toxicities were observed. Of ten evaluable patients, five had progressive disease in 4 to 8 weeks; three had stable disease for 12 to 21 weeks; one has had a minor response for 13 weeks; and one has had a complete resolution of tumor for 150 + weeks. IFN-_ser appears to have activity in human glioma and is well tolerated at this dosage and schedule.