The growth strategy of the Gram-positive rod

Abstract Bacteria grow by enlarging their envelope in such a way that osmotic pressure does not normally cause physical rupture. The strategy of Bacillus subtilis for both cylindrical elongation and pole formation is now substantially defined. Side-wall growth takes place by laying down new peptidoglycan, which is then displaced outwards, stretched and discarded; cross walls are laid down in the absence of stress, and then stretched and bulged outward as the septum is split and the pole is formed.     

A statistical power analysis of the 'internal reference' technique for comparing growth and growth depensation of tilapia strains

Experiments were conducted to compare the growth and growth compensation of three strains of juvenile Oreochromis niloticus . Ten full sib families (10 replicates) per strain were split and grown under crowded and uncrowded conditions for 3 weeks (the treatment). Both treatments were then grown an additional 12 weeks under less crowded conditions (the compensation). Standard length measurements were made at the end of crowding and the end of compensation. Each replicate included a size-matched control population of a fourth (red) reference strain. ANCOVA with the reference strain used as a cofactor revealed significant strain effects on specific growth throughout the experiment. The reference strain removed most of the random among-replicate error variance as shown by an increase of r 2 from 0.06 to 0.91 when it was included in the statistical models. If the reference fish had not been used, approximately 450 replicate families would have been needed to achieve the sensitivity of the present experiment (a difference of 7% among strains significant at P =0.05). We conclude that the CLSU strain grows significantly more slowly than the Israel and NIFI strains under the experimental conditions, that the crowding effect was essentially eliminated after 12 weeks of compensation, and that the reference strain greatly improved the resolution of the strain-testing experiment.     

Quantitative detection of C-reactive protein using phosphocholine-labelled enzyme or microspheres

C-reactive protein (CRP) is a positive, acute-phase protein. Plasma levels rise dramatically in response to tissue injury or inflammation and fall rapidly after recovery or treatment. Antibody-based human CRP test systems do not readily detect CRP from other animals due to the species specificity of antibodies directed against human CRP. Thus, generic systems for CRP detection, based solely on the interaction between CRP and phosphocholine (PC), have been developed. PC-bovine serum albumin (PC-BSA) conjugates were produced and either labeled with horseradish peroxidase to facilitate CRP detection in a CRP enzyme-linked sorbent assay (ELSA) or coupled to carboxy-modified microspheres to facilitate the nonenzymatic, turbidimetric detection of CRP. The CRP-ELSA is a competitive assay, where the total assay time is 45 min, the assay sensitivity is 1.06 mg/L CRP, and the dynamic range of the assay is 0-500 mg/L. When PC-BSA conjugate is covalently coupled to carboxylated microspheres, agglutination occurs in the presence of CRP, the extent of which depends on the quantity of CRP present in the sample. Total assay time is 5 min with a dynamic range of 25-500 mg/L. Both assay formats are capable of accurately detecting human CRP and the CRP-ELSA can detect canine CRP as a disease state indicator.     

Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.     

Chemical complementation: small-molecule-based genetic selection in yeast

Protein and metabolic engineering would greatly benefit from a general system linking the presence of a small molecule to the power of genetic selection. We use nuclear receptors to link the survival of Saccharomyces cerevisiae to the presence of small molecules through genetic selection, extending classical genetic complementation to a new "chemical complementation." In this system the Gal4 DNA-binding domain is fused to ligand-binding domains from two nuclear receptors, expressed in the strain PJ69-4A, and grown on plates containing known ligands for the receptors. Yeast survive on selective plates only in the presence of a nuclear receptor and the corresponding ligand. Mutagenesis can increase the sensitivity of chemical complementation. This system may be extended to engineer nuclear receptors for practically any small molecule through directed evolution coupled to genetic selection, and for performing metabolic engineering in yeast.     

Measuring kinesin's first step

A variety of models have recently emerged to explain how the molecular motor kinesin is able to maintain processive movement for over 100 steps. Although these models differ in significant features, they all predict that kinesin's catalytic domains intermittently separate from each other as the motor takes 8-nm steps along the microtubule. Furthermore, at some point in this process, one molecule of ATP is hydrolyzed per step. However, exactly when hydrolysis and product release occur in relation to this forward step have not been established. Furthermore, the rate at which this separation occurs as well as the speed of motor stepping onto and release from the microtubule have not been measured. In the absence of this information, it is difficult to critically evaluate competing models of kinesin function. We have addressed this issue by developing spectroscopic probes whose fluorescence is sensitive to motor-motor separation or microtubule binding. The kinetics of these fluorescence changes allow us to directly measure how fast kinesin steps onto and releases from the microtubule and provide insight into how processive movement is maintained by this motor.     

On morphometric measurement of oxygen diffusing capacity in middle ear gas exchange.

An accurate mathematical model of transmucosal gas exchange is prerequisite to understanding middle ear (ME) physiology. Current models require experimentally measured gas species time constants for all extant conditions as input parameters. However, studies on pulmonary gas exchange have shown that a morphometric model that incorporates more fundamental physiochemical and anatomic parameters accurately simulates transport from which the species time constants can be derived for all extant conditions. Here, we implemented a variant of that model for ME gas exchange that requires the measurement of diffusional length (tau) for the ME mucosa. That measure contributes to the mucosal diffusing capacity and reflects the resistance to gas flow between air space and capillary. Two methods for measuring tau have been proposed: linear distance between the air-mucosal boundary and capillary and the harmonic mean of all contributing pathway lengths. Oxygen diffusing capacity was calculated for different ME mucosal geometries by using the two tau measures, and the results were compared with those predicted by a detailed, two-dimensional finite element analysis. Predictive accuracy was improved by incorporating the harmonic tau measure, which captures important information regarding variations in capillary shape and distribution. However, compared with the oxygen diffusing capacity derived from the finite element analysis, both measures yielded nonlinear, positively biased estimates. The morphometric techniques underestimate diffusion length by failing to account for the curvilinear gas flow pathways predicted by the finite element model.