Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography

As part of a study to purify the internal domain of HER2 (ICD) from recombinant expression, through metal immobilised affinity chromatography (IMAC), we encountered a contaminant, SlyD, a 29 kDa native E. coli protein. SlyD is a recurrent contaminant, with a histidine rich domain enabling binding to IMAC columns and thus co-elution with the target protein. Research has been carried out on this protein and its purification, however, no work mentions how to treat it as a true contaminant or describe procedures to isolate it from target proteins. In this report, we described a two-step chromatographic method for the purification of ICD, including IMAC as a capture step and size exclusion chromatography (SEC) as a polishing step. IMAC allowed us to purify ICD from bacterial crude with SlyD co-eluting. SEC then allowed us to resolve ICD from SlyD and achieve a purity greater than 95% for ICD. However, this method has been developed to accommodate any protein whose molecular weight is different enough from SlyD to be separated by SEC.     

Coaxial lines for multiphase power distribution

A coaxial cable can be used to reduce the magnetic and electric fields that extend into environments in the vicinity of transmission lines and distribution lines and in-house or building wiring for power distribution systems. The use of the coaxial geometry may prove useful in cases where there are environmental concerns with respect to health effects and in cases where there is a need to run high-speed data communications in close proximity to power distribution systems. © 1996 Wiley-Liss, Inc.     

The change in the rate of muscle protein metabolism of rats from weaning to 90 days of age

1. Protein synthesis, net deposition and breakdown was studied in the gastrocnemius muscles of growing rats between weaning and 90 days of age. 2. Fractional protein synthetic rates declined from 30.02% at 25 days to 7.41% at 90 days. 3. The rate of protein degradation follows a similar pattern to that of protein synthesis. A linear relationship was found. 4. The break in the growth curve between 30 and 31 days was also observed in protein metabolism.     

Buffers for the reconstitution of aspartate aminotransferase

The cofactor activation of the apoenzyme of pig heart cytosolic aspartate aminotransferase was studied in various buffers. Cationic buffers are shown to allow maximal reconstitution in the pH range of 5.0 to 9.0. Anionic buffers made up of mono- and dicarboxylates are found to affect reconstitution in a pH-dependent manner. At low pH, the carboxylates strongly inhibit reconstitution, but at high pH, they show less effect. In contrast, the more potent inhibitor Pi shows the opposite pH profile. Dicarboxylates are considerably more inhibitory than monocarboxylates. Substantial protection against inhibition by a number of carboxylates may be achieved by the addition of sodium chloride.     

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

The influence of depth,site exposure and season on the intensity of iceberg scouring in nearshore Antarctic waters

Ice scour disturbance has a significant effect on the physical and biological characteristics of polar benthos. A series of grids, each consisting of 25 markers, were deployed along depth transects and replicated at two contrasting study sites at Adelaide Island, West Antarctic Peninsula. Markers were surveyed and replaced every 3 months for 2 years in order to assess the frequency and intensity of iceberg impacts. Depth, site, season and year were all highly significant factors influencing ice scouring frequency. We observed a high variation in the duration of winter fast ice between sites and years, which had a marked effect on ice scouring frequency. The ecological effects of the disturbance regime are likely to include depth zonation of benthic assemblages, patchiness of communities at varying stages of recovery and the near denudation of sessile fauna in the shallow subtidal.

Tötung von Hefen durch Strahlungen des menschlichen Körpers

Zusammenfassung Bei vier Personen, zwei männlichen und zwei weiblichen, konnte eine Emanation aus den Fingerspitzen festgestellt werden, welche eine bestimmte Hefenart (Saccharomyces Mycoderma punctisporus Guill.) in kurzer Zeit tötete.Bei allen vier Personen konnte diese Strahlung auf pathologische Zustände zurückgeführt werden.Die Strahlung ging nicht durch Glas, wohl aber durch Quarz und ist daher sehr wahrscheinlich ultraviolett.Chemisch hergestelltes Oxycholesterin verursacht in Emulsion mit Wasser ebenfalls Tötung der Hefe durch Quarz hindurch. Oxycholesterin ist ein Bestandteil des aus den Talgdrüsen ausgeschiedenen Fettes.Der Snyder Research Foundation, durch deren finanzielle Unterstützung diese Arbeit ermöglicht wurde, sei auch an dieser Stelle bestens gedankt.     

Effect of bradykinin on airway neural responses in vitro.

We investigated the effects of bradykinin (BK) on airway excitatory nonadrenergic noncholinergic (e-NANC) and cholinergic nerves in vitro. Neural responses were elicited by electrical field stimulation in guinea pig airways in vitro before and after the addition of BK (10(-10)-10(-7) M). Captopril (10(-5) M) and phosphoramidon (10(-6) M) were added to prevent degradation of BK, and all neural responses were measured in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M). BK potentiated e-NANC responses in bronchi in a concentration-dependent manner (10(-10)-10(-7) M) without changing concentration-response curves to exogenously applied substance P (10(-10)-10(-5) M). BK significantly potentiated e-NANC neural constrictor responses by 22 +/- 7% at 10(-8) M (mean +/- SE, n = 5, P < 0.05) and 32 +/- 7% at 10(-7) M (n = 8, P < 0.01), compared with changes in time-matched control tissues (7 +/- 2%, n = 8). The potentiation of e-NANC responses by BK was abolished by pretreatment with a specific B2-receptor antagonist, HOE 140 (10(-7) M). Cholinergic constrictor responses elicited to electrical field stimulation were not affected by the addition of BK (up to 10(-7) M). These results suggest that BK potentiates e-NANC bronchoconstrictor responses prejunctionally via a B2-receptor.