Quantitative Determination of Gap Junctional Permeability in the Lens Cortex

We have developed a simple dye transfer method, which allows the gap junction permeability of lens fiber cells to be quantified. Two fixable fluorescent dyes (Lucifer yellow and rhodamine-dextran) were introduced into peripheral lens fiber cells via mechanical damage induced by removing the lens capsule. After a defined incubation period, lenses were fixed, sectioned, and the distribution of the dye recorded using confocal microscopy. Rhodamine-dextran and Lucifer yellow both labeled the extracellular space between fiber cells and the cytoplasm of fiber cells that had been damaged by capsule removal. For the gap junctional permeable dye Lucifer yellow, however, labeling was not confined to the damaged cells and exhibited intercellular diffusion away from the damaged cells. The extent of dye diffusion was quantified by collecting radial dye intensity profiles from the confocal images. Effective diffusion coefficients (D _eff ) for Lucifer yellow were then calculated by fitting the profiles to a series of model equations, which describe radial diffusion in a sphere. D _eff is the combination of dye diffusion through the cytoplasm and through gap junction channels. To calculate the gap junctional permeability (P _j ) an estimate of the cytoplasmic diffusion coefficient (D _cyt = 0.7 × 10⁻⁶ cm²/sec) was obtained by observing the time course of dye diffusion in isolated elongated fiber cells loaded with Lucifer yellow via a patch pipette. Using this approach, we have obtained a value for P _j of 31 × 10⁻⁵ cm/sec for fiber-fiber gap junctions. This value is significantly larger than the value of P _j of 4.4 × 10⁻⁶ cm/sec reported by Rae and coworkers for epithelial-fiber junctions (Rae et al., 1996. J. Membrane Biol. 150:89–103), and most likely reflects the high abundance of gap junctions between lens fiber cells. Received: 1 December 1998/Revised: 22 February 1999     

Fusarium graminearum Tri12p influences virulence to wheat and trichothecene accumulation

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.     

Molecular portrait of lens gap junction protein MP70

A 70-kDa membrane protein (MP70) is a component of the lens fiber gap junctions. Its membrane topology and its N-terminal sequence are similar to those of the connexin family of proteins. Some features of MP70 containing fiber gap junctions are, however, distinct from gap junctions in other mammalian tissues: (i) Lens connexons form crystalline arrays only after cleavage of junctional proteins in vitro. These hexagonal arrays have a periodicity of 13.6 nm which is significantly larger than the 8- 9-nm spacing of liver and heart gap junctions. (ii) Lens fiber gap junctions dissociate in low concentrations of nonionic detergent and this provides an avenue to purify MP70 directly from a membrane mixture. Isolated MP70 in the form of 17 S structures has an appearance consistent with connexon pairs. (iii) The C-terminal half of MP70 is cleaved in situ by a lens endogenous calcium-dependent protease. The processed from MP38 remains in the membrane and is abundant in the central region of the lens. A testable hypothesis for MP70 function is presented.     

Effects of Piper hispidinervum on spermatogenesis and histochemistry of ovarioles of Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3^rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest.     

Amyloid fibril formation from full-length and fragments of amylin

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.     

Purification and characterization of the creatine transporter expressed at high levels in HEK293 cells

The bovine creatine transporter (CreaT) has been purified from membranes of HEK293 cells stably expressing high levels of the transporter. Membranes were solubilized with decyl maltoside and the CreaT was purified (90% pure) by affinity chromatography on wheat germ agglutinin (WGA)-Sepharose and gel-filtration. The CreaT was shown to be an approximately 70 kDa glycoprotein by SDS-polyacrylamide gel electrophoresis and Western blotting. Identification of the CreaT was confirmed by sequencing tryptic peptides by mass spectrometry. Laser light scattering showed the majority of the CreaT to be present as a 224 kDa species. Additional purification was obtained when the Creat was eluted from the WGA column and purified by gel-filtration in Fos-choline 12 instead of decyl maltoside, followed by a second WGA affinity step to exchange the detergent for sodium cholate. This resulted in a 30-fold purification (95% purity) of the approximately 70kDa CreaT, with a yield of 15%. From this, it is estimated that the CreaT comprises approximately 3% of total HEK293-CreaT membrane protein. Gel-filtration showed the transporter to migrate with an apparent molecular mass of 210 kDa. Circular dichroism showed a predominantly alpha-helical structure, consistent with the 12 transmembrane domains predicted for the transporter. This work has enabled the purification of the CreaT in amounts ( approximately 100 microg) that make it feasible to consider structural studies of a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter family.     

Mechanisms for cell activation and its consequences for biorheology and microcirculation: Multi-organ failure in shock

Activation of cells in the vascular compartment causes profound alteration of cell rheological properties with impairment of the microcirculation and initiation of inflammatory reactions. Many cardiovascular diseases have been shown to be associated with cell activation and inflammation. While this situation offers the opportunity for new interventions against the deleterious effects of cell activation, there is the need for a better understanding of the mechanisms that lead to cell activation in the first place. We review here several mechanisms for cell activation in the circulation. We show that in shock, a condition associated with severe forms of cell activation, humoral cell activation factors can be detected in plasma. Further analysis indicates that the source of these humoral activators may be due to the action of pancreatic digestive enzymes in the intestine. Ischemia may serve to open the intestinal brush border and permit entry of pancreatic enzymes into the wall of the intestine to initiate self digestion. In this process low molecular weight but potent cell activators are produced which may escape via the intestinal circulation and the lymphatics into the general circulation. Inhibition of pancreatic enzymes in the lumen of the intestine leads to complete attenuation of humoral activator production as well as many of the deleterious sequelae that accompany shock, such as inflammation and multi-organ failure. We outline a method to carry out biochemical isolation of the cell activators derived from pancreatic enzymes. This analysis shows that there are multiple species of cell activators above and beyond currently known species, many of which have molecular weights below 3000 Da. Identification of the mechanisms that lead to cell activation is an important part to understand the mechanisms that lead to alterations of rheological properties of blood cells in disease and dysfunction of the endothelium and parenchymal cells. Our current evidence suggests that pancreatic digestive enzymes and tissue enzymes may play a central role in humoral activator production.     

PRACTICAL STEREOLOGICAL METHODS FOR MORPHOMETRIC CYTOLOGY

Stereological principles provide efficient and reliable tools for the determination of quantitative parameters of tissue structure on sections. Some principles which allow the estimation of volumetric ratios, surface areas, surface-to-volume ratios, thicknesses of tissue or cell sheets, and the number of structures are reviewed and presented in general form; means for their practical application in electron microscopy are outlined. The systematic and statistical errors involved in such measurements are discussed.