Lung overexpansion increases pulmonary microvascular protein permeability in young lambs

To study the effects of inflation pressure and tidal volume (VT) on protein permeability in the neonatal pulmonary microcirculation, we measured lung vascular pressures, blood flow, lymph flow (QL), and concentrations of protein in lymph (L) and plasma (P) of 22 chronically catheterized lambs that received mechanical ventilation at various peak inflation pressures (PIP) and VT. Nine lambs were ventilated initially with a PIP of 19 +/- 1 cmH2O and a VT of 10 +/- 1 ml/kg for 2-4 h (base line), after which we overexpanded their lungs with a PIP of 58 +/- 3 cmH2O and a VT of 48 +/- 4 ml/kg for 4-8 h. QL increased from 2.1 +/- 0.4 to 13.9 +/- 5.0 ml/h. L/P did not change, but the ratio of albumin to globulin in lymph relative to the same ratio in plasma decreased, indicating altered protein sieving in the pulmonary microcirculation. Seven other lambs were mechanically ventilated for 2-4 h at a PIP of 34 +/- 1 cmH2O and a VT of 23 +/- 2 ml/kg (base line), after which their chest and abdomen were bound so that PIP increased to 54 +/- 1 cmH2O for 4-6 h without a change in VT. QL decreased on average from 2.8 +/- 0.6 to 1.9 +/- 0.3 ml/h (P = 0.08), and L/P was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

1.56 Å structure of mature truncated human fibroblast collagenase

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 Å resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded β-sheet and three α-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase. © 1994 Wiley-Liss, Inc.     

The role of touch,pressure and nociceptive mechanoreceptors of the leech in unrestrained behaviour

1. The maximum force exerted against an isometric force transducer by 6 leeches weighing 2.6–3.7 g, as they squeezed through apertures of different widths varied inversely with aperture width.
2. T cells in the leech skin code for velocity of indentation, not pressure or displacement. The frequency with which T cells fire is best described by two log functions, one for low, another for fast indentations. T cells responded to indentation velocities down to 10 μms^?1.
3. The average threshold pressure for 5 P cells was 150 kPa and for 5 N cells was 521 kPa.
4. We conclude from these data that when leeches explore their mechanical environment and initiate contact with external objects, the threshold pressure for N cells is rarely crossed. Of the three classes of mechanoreceptor, T cells are the main modality through which leeches obtain contact information, though P cells may occasionally be recruited for local pressure peaks.

     

A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis

Bacillus thuringiensis var. israelensis (BTI), serotype 14, which produces parasporal crystals toxic to certain dipteran larvae, was analyzed by agarose gel electrophoresis and found to contain a complex plasmid array. Eight plasmids were detected, with approximate sizes of 3.3, 4.2, 4.9, 10.6, 68, 75, 105, and 135 MDa, as well as a plasmidlike linear DNA element of ~10 MDa. Partially cured mutants of BTI implicated the 75-MDa plasmid in crystal production. Fifteen independently isolated acrystalliferous (Cry^?) mutants were found to lack this plasmid. In plasmid transfer experiments, several of the BTI plasmids transferred into a plasmid-free, Cry^? BTI recipient, but only transfer of the 75-MDa plasmid converted the recipient to crystal toxin production. The presence or absence of mosquito-toxic activity in all Cry⁺ and Cry^? variants of BTI was confirmed by bioassay of sporulated cultures against larvae of Aedes aegypti. Southern blot analyses revealed that in one unusual Cry⁺ variant in which no 75-MDa plasmid band was detectable, plasmid sequences were still present, possibly integrated into the chromosome. The 75-MDa plasmid could also apparently recombine with the 68-MDa plasmid, to which it was partially homologous.     

Physical mapping of a plasmid from Bacillus megaterium by restriction endonuclease cleavage.

A 4-Mdalton plasmid from Bacillus megaterium strain 216 has been physically mapped by restriction endonuclease digestion. A combination of single and double digests with seven restriction enzymes, together with a terminal labeling procedure, has produced a physical map containing 21 apparently unique cleavage sites. The data are most consistent with the view that this plasmid does not contain extensive variability in sequence.     

Novel surface expression of reticulocalbin 1 on bone endothelial cells and human prostate cancer cells is regulated by TNF-alpha

An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-alpha.     

Role of fibroblast growth factor receptors 1 and 2 in the metanephric mesenchyme

To determine the importance of fibroblast growth factor receptors (fgfrs) 1 and 2 in the metanephric mesenchyme, we generated conditional knockout mice (fgfr(Mes-/-)). Fgfr1(Mes-/-) and fgfr2(Mes-/-) mice develop normal-appearing kidneys. Deletion of both receptors (fgfr1/2(Mes-/-)) results in renal aplasia. Fgfr1/2(Mes-/-) mice develop a ureteric bud (and occasionally an ectopic bud) that does not elongate or branch, and the mice do not develop an obvious metanephric mesenchyme. By in situ hybridization, regions of mutant mesenchyme near the ureteric bud(s) express Eya1 and Six1, but not Six2, Sall1, or Pax2, while the ureteric bud expresses Ret and Pax2 normally. Abnormally high rates of apoptosis and relatively low rates of proliferation are present in mutant mesenchyme dorsal to the mutant ureteric bud at embryonic day (E) 10.5, while mutant ureteric bud tissues undergo high rates of apoptosis by E11.5. Thus, fgfr1 and fgfr2 together are critical for normal formation of metanephric mesenchyme. While the ureteric bud(s) initiates, it does not elongate or branch infgfr1/2(Mes-/-) mice. In metanephric mesenchymal rudiments, fgfr1 and fgfr2 appear to function downstream of Eya1 and Six1, but upstream of Six2, Sall1, and Pax2. Finally, this is the first example of renal aplasia in a conditional knockout model.