Hydrodynamic thickening of lubricating fluid layer beneath sliding mesothelial tissues

The delicate mesothelial surfaces of the pleural space and other serosal cavities slide relative to each, lubricated by pleural fluid. In the absence of breathing motion, differences between lung and chest wall shape could eventually cause the lungs and chest wall to come into contact. Whether sliding motion keeps lungs and chest wall separated by a continuous liquid layer is not known. To explore the effects of hydrodynamic pressures generated by mesothelial sliding, we measured the thickness of the liquid layer beneath the peritoneal surface of a 3-cm disk of rat abdominal wall under a normal stress of 2 cm H2O sliding against a glass plate rotating at 0-1 rev/s. Thickness of the lubricating layer was determined microscopically from the appearance of fluorescent microspheres adherent to the tissue and glass. Usually, fluid thickness near the center of the tissue disk increased with the onset of glass rotation, increasing to 50-200 microm at higher rotation rates, suggesting hydrodynamic pumping. However, thickness changes often differed substantially among tissue samples and between clockwise and counter-clockwise rotation, and sometimes thickness decreased with rotation, suggesting that topographic features of the tissue are important in determining global hydrodynamic effects. We conclude that mesothelial sliding induces local hydrodynamic pressure gradients and global hydrodynamic pumping that typically increases the thickness of the lubricating fluid layer, moving fluid against the global pressure gradient. A similar phenomenon could maintain fluid continuity in the pleural space, reducing frictional force and shear stress during breathing.     

Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 × 10² copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.     

A gene expression profile of Alzheimer's disease.

Postmortem analysis of brains of patients with Alzheimer's disease (AD) has led to diverse theories about the causes of the pathology, suggesting that this complex disease involves multiple physiological changes. In an effort to better understand the variety and integration of these changes, we generated a gene expression profile for AD brain. Comparing affected and unaffected brain regions in nine controls and six AD cases, we showed that 118 of the 7050 sequences on a broadly representative cDNA microarray were differentially expressed in the amygdala and cingulate cortex, two regions affected early in the disease. The identity of these genes suggests the most prominent upregulated physiological correlates of pathology involve chronic inflammation, cell adhesion, cell proliferation, and protein synthesis (31 upregulated genes). Conversely, downregulated correlates of pathology involve signal transduction, energy metabolism, stress response, synaptic vesicle synthesis and function, calcium binding, and cytoskeleton (87 downregulated genes). The results support several separate theories of the causes of AD pathology, as well as add to the list of genes associated with AD. In addition, approximately 10 genes of unknown function were found to correlate with the pathology.     

Relative strengths of the chest wall muscles

We hypothesized that during maximal respiratory efforts involving the simultaneous activation of two or more chest wall muscles (or muscle groups), differences in muscle strength require that the activity of the stronger muscle be submaximal to prevent changes in thoracoabdominal configuration. Furthermore we predicted that maximal respiratory pressures are limited by the strength of the weaker muscle involved. To test these hypotheses, we measured the pleural pressure, abdominal pressure (Pab), and transdiaphragmatic pressure (Pdi) generated during maximal inspiratory, open-glottis and closed-glottis expulsive, and combined inspiratory and expulsive maneuvers in four adults. We then determined the activation of the diaphragm and abdominal muscles during selected maximal respiratory maneuvers, using electromyography and phrenic nerve stimulation. In all subjects, the Pdi generated during maximal inspiratory efforts was significantly lower than the Pdi generated during open-glottis expulsive or combined efforts, suggesting that rib cage, not diaphragm, strength limits maximal inspiratory pressure. Similarly, at high lung volumes, the Pab generated during closed-glottis expulsive efforts was significantly greater than that generated during open-glottis efforts, suggesting that the latter pressure is limited by diaphragm, not abdominal muscle, strength. As predicted, diaphragm activation was submaximal during maximal inspiratory efforts, and abdominal muscle activation was submaximal during open-glottis expulsive efforts at midlung volume. Additionally, assisting the inspiratory muscles of the rib cage with negative body-surface pressure significantly increased maximal inspiratory pressure, whereas loading the rib cage muscles with rib cage compression decreased maximal inspiratory pressure. We conclude that activation of the chest wall muscles during static respiratory efforts is determined by the relative strengths and mechanical advantage of the muscles involved.     

Two novel polymorphic sequences in the glucocerebrosidase gene region enhance mutational screening and founder effect studies of patients with Gaucher disease

Gaucher disease, an inherited glycolipid storage disorder, is caused by a deficiency of the catabolic enzyme glucocerebrosidase (EC 3.2.1.45). The gene for human glucocerebrosidase is located on chromosome 1q21 and has a highly homologous pseudogene situated 16 kb downstream. We report two novel polymorphic sequences in the glucocerebrosidase gene region: the first consists of a variable number of dinucleotide (CT) repeats located 3.2 kb upstream from the glucocerebrosidase gene, and the second is a tetranucleotide (AAAT) repeat found between the glucocerebrosidase gene and its pseudogene, 9.8 kb downstream from the functional gene. These polymorphic sequences, along with a previously reported PvuII polymorphism in intron 6 of the glucocerebrosidase gene, were analyzed in patients with Gaucher disease (n=106) and in two normal control populations, one of Ashkenazi Jewish ancestry (n=72) and the second comprising non-Jewish individuals (n=46). In these samples, strong linkage disequilibrium was found between mutations N370S, c.84–85insG, and R463C and specific haplotypes; no significant linkage disequilibrium was found when examining haplotypes of patients with the L444P mutation. Studies of these polymorphic sites in several instances also led to the recognition of genotyping errors and the identification of unusual recombinant alleles. These new polymorphic sites provide additional tools for mutational screening and founder effect studies of Gaucher disease. Received: 5 December 1998 / Accepted: 14 January 1999