Characterization and purification of a Na+/Ca2+ exchanger from an archaebacterium

The superfamily of cation/Ca(2+) exchangers includes both Na(+)/Ca(2+) exchangers (NCXs) and Na(+)/Ca(2+),K(+) exchangers (NCKX) as the families characterized in most detail. These Ca(2+) transporters have prominent physiological roles. For example, NCX and NCKX are important in regulation of cardiac contractility and visual processes, respectively. The superfamily also has a large number of members of the YrbG family expressed in prokaryotes. However, no members of this family have been functionally expressed, and their transport properties are unknown. We have expressed, purified, and characterized a member of the YrbG family, MaX1 from Methanosarcina acetivorans. MaX1 catalyzes Ca(2+) uptake into membrane vesicles. The Ca(2+) uptake requires intravesicular Na(+) and is stimulated by an inside positive membrane potential. Despite very limited sequence similarity, MaX1 is a Na(+)/Ca(2+) exchanger with kinetic properties similar to those of NCX. The availability of a prokaryotic Na(+)/Ca(2+) exchanger should facilitate structural and mechanistic investigations.     

Risk Factors for Severe Neutropenia following Intra-Arterial Chemotherapy for Intra-Ocular Retinoblastoma

Purpose

Intra-arterial chemotherapy is a promising strategy for intra-ocular retinoblastoma. Neutropenia is the most commonly encountered systemic toxicity and in this study we aimed to determine the risk factors associated with the development of severe (≥grade 3) neutropenia.

Methods

Retrospective review of 187 evaluable cycles of melphalan-containing intra-arterial chemotherapy from the first three cycles administered to 106 patients with intra-ocular retinoblastoma from May 2006 to June 2011. Cycles were considered to be evaluable if (1) blood count results were available in the 7 to 14 days post-treatment interval and (2) concurrent intravenous chemotherapy was not administered. Toxicity was assessed via the Common Terminology Criteria for Adverse Events version 4.0.

Results

54 cycles (29%) were associated with grade 3 (n = 43) or grade 4 (n = 11) neutropenia. Multivariate stepwise logistic regression revealed that a higher melphalan dose (>0.40 mg/kg) was significantly associated with severe neutropenia during all 3 cycles (odds ratio during cycle one 4.11, 95% confidence interval 1.33–12.73, p = 0.01), but the addition of topotecan and/or carboplatin were not. Prior treatment with systemic chemotherapy was not associated with severe neutropenia risk in any analysis.

Conclusions

Intra-arterial melphalan-based chemotherapy can cause severe neutropenia, especially when a dose of greater than 0.40 mg/kg is administered. Further study with a larger sample may be warranted.     

F-Spondin Deficient Mice Have a High Bone Mass Phenotype

F-spondin is a pericellular matrix protein upregulated in developing growth plate cartilage and articular cartilage during osteoarthritis. To address its function in bone and cartilage in vivo, we generated mice that were deficient for the F-spondin gene, Spon1. Spon1 ^{− /−} mice were viable and developed normally to adulthood with no major skeletal abnormalities. At 6 months, femurs and tibiae of Spon1 ^{− /−} mice exhibited increased bone mass, evidenced by histological staining and micro CT analyses, which persisted up to 12 months. In contrast, no major abnormalities were observed in articular cartilage at any age group. Immunohistochemical staining of femurs and tibiae revealed increased levels of periostin, alkaline phosphate and tartrate resistant acid phosphatase (TRAP) activity in the growth plate region of Spon1 ^{− /−} mice, suggesting elevated bone synthesis and turnover. However, there were no differences in serum levels of TRAP, the bone resorption marker, CTX-1, or osteoclast differentiation potential between genotypes. Knockout mice also exhibited reduced levels of TGF-β1 in serum and cultured costal chondrocytes relative to wild type. This was accompanied by increased levels of the BMP-regulatory SMADs, P-SMAD1/5 in tibiae and chondrocytes. Our findings indicate a previously unrecognized role for Spon1 as a negative regulator of bone mass. We speculate that Spon1 deletion leads to a local and systemic reduction of TGF-β levels resulting in increased BMP signaling and increased bone deposition in adult mice.     

Can Honey Bees Learn the Removal of a Stimulus as a Conditioning Cue?

Experiments investigated a Pavlovian conditioning situation where the presence and absence of the stimulus are reversed temporally with respect to the presentation of a reward. Instead of a conditioned stimulus (e.g. odor) signaling the presence of a reward, the stimulus (e.g. odor) is present in the environment except just prior to the presence of the reward. Thus, the absence of the stimulus, or offset of the stimulus (e.g. absence of odor), serves as a conditioned stimulus and is the reward cue. Honey bees (Apis mellifera) were used as a model invertebrate system, and the proboscis‐conditioning paradigm was used as the test procedure. Using both simple Pavlovian conditioning and discrimination‐learning protocols, animals learned to associate the onset of an odor as conditioned stimuli when paired with a sucrose reward. They could also learn to associate the onset of a puff of air with a sucrose reward. However, bees could not associate the offset of an order stimulus with the presentation of a sucrose reward in either a simple conditioning or a discrimination‐learning situation. These results support the model that a very different cognitive architecture is used by invertebrates to deal with certain environmental situations, including signaled avoidance.     

Microflora and condition of flood-damaged grains in Manitoba,Canada

Microfloral and other changes which occurred in farm-stored grains at, below and above the maximum water level were documented after the Red River Valley flood of spring 1979 in southern Manitoba, Canada. Results of flood-damage simulation studies carried out in the laboratory on wheat, rapeseed and flax generally confirmed those from the field. In laboratory simulation studies with flooded grains in tanks a 2.5–10.0 cm thick crust of sprouted seeds developed just above the maximum water level. Migration of moisture occurred upwards from the base of the crust and maximum temperatures just beneath the crust. Beneath the crust was a horizon of putrid seeds of zero germination, and above the crust a horizon of visible post-harvest molds, e.g.Aspergillus flavus andPenicillium spp. and harvest molds, e.g.Trichothecium roseum. Penicillium verrucosum var.cyclopium occurred throughout the profile, whereasAlternaria alternata, Wallemia sebi andAspergillus glaucus group species were confined to upper horizons because of low seed moisture andA. flavus and A. versicolor to lower horizons of high moisture. Aflatoxins B₁ at 426 ppb and B₂ at 34 ppb were the only mycotoxins detected in either study, and occurred in a partially filled granary of flax which had been affected by flood waters. Pressure effects produced by the expanding seeds such as increase in height of grain mass and splitting of seams of granaries were evident in both studies. Rounded or slippery seeds, e.g. peas, flax, were associated usually with vertical pressure effects whereas irregularly-shaped seeds, e.g. wheat with lateral pressure effects. It is hoped that this work will provide a better understanding of the processes involved when grain is damaged by flood waters and assist in the development of improved salvage procedures.Contribution No. 942, Agriculture Canada Research Station, 195 Dafoe Road, Winnipeg, Manitoba, R3T 2M9.     

A NEW CHEMICAL METHOD FOR QUANTIFYING MELANIN1

—A direct chemical method for the estimation of absolute amounts of synthetic melanin is established by the use of sodium borohydride, a mild reducing agent for the ketonic or aldehyde carbonyl group. Sodium borohydride not only solubilized the melanins obtained from various precursors, but it also restored aromatic characteristics in the absorption spectra. The solubilized melanin has also been used with a radio-assay method for quantification of synthetic melanin. With this method of solubilization, the presence of lipids or proteins does not alter the linearity of the assay. The limiting amount of melanin detected by this method is 4-5 μg/ml of the final solution.     

A METHOD FOR THE RAPID DIALYSIS OF LARGE VOLUMES OF PROTEIN SOLUTIONS

A method is described by which moderately large quantities of protein solutions can be dialyzed relatively free of salts within 12 to 24 hours. The use of "Visking" tubes instead of collodion bags is recommended, for speed of dialysis as well as ease of manipulation.     

ELECTROKINETIC PHENOMENA : I. THE ADSORPTION OF SERUM PROTEINS BY QUARTZ AND PARAFFIN OIL

1. The effect of human and rabbit sera on the cataphoretic mobility of glass and quartz particles, and of paraffin oil droplets, was studied in serum dilutions (with 0.85 per cent NaCl) from 1:50 to 1:1,000,000, over a pH range of 3.6 to 9.3. 2. Under the conditions described, these various types of particles adsorbed protein partially or completely from the most dilute solution giving these particles electrokinetic properties characteristic of certain proteins, probably here those of serum albumin. 3. Quartz particles and paraffin oil droplets both have an isoelectric point between 4.7 and 4.8 in a 1:50 serum dilution. 4. The biological importance of these findings is discussed. 5. A non-polarizable electrode composed of See PDF for Structure is described for use with cataphoresis cells.     

THE INFLUENCE OF SIZE,SHAPE AND CONDUCTIVITY OF MICROSCOPICALLY VISIBLE PARTICLES ON CATAPHORETIC MOBILITY

The electrophoretic mobility of microscopically visible particles is independent of size, shape and conductivity of the particle within the limits of the experimental error. This is valid for extreme variations in size, shape and conductivity.