Enhanced disease resistance conferred by expression of an antimicrobial magainin analog in transgenic tobacco.

Magainins are a group of short peptides originally isolated from frog skin and thought to function as a natural defense mechanism against infection due to their antimicrobial properties. The engineered magainin analog peptide Myp30 was found to inhibit spore germination of the oomycete, Peronospora tabacina (Adam) in vitro, and the growth of a bacterial pathogen Erwinia carotovora subsp. carotovora (Jones). Transgenic tobacco (Nicotiana tabacum L.) plants expressing Myp30 were evaluated for resistance to these pathogens. The expression of the peptide only to an extracellular location resulted in significant reduction in sporulation and lesion size due to P. tabacina infection. A significant increase in resistance to the bacterial pathogen was also observed regardless of the targeting location of the peptide.

Age-related changes in albumin-excluded volume fraction.

The space available to large macromolecules, such as albumin and globulin, is less than the total interstitial fluid volume due to the dense matrix formed by the interstitial ground substance. Changes in excluded volume are likely to indicate changes in the composition of the matrix. Sprague-Dawley rats were anesthetized with sodium pentobarbital. Serum, mesenteric tissue, and peritoneal fluid samples were obtained. Albumin contents were determined by microrod electrophoresis. Serum and mesenteric tissue chloride concentrations were measured by the coulometric-amperometric method. Serum and mesenteric tissue sample chloride concentrations were not significantly different, suggesting that this loose connective tissue is composed almost entirely of extracellular matrix. Matrix hydration decreased with a regression slope of -0.014 (microgram tissue water/microgram tissue dry wt)/10 days. Serum and tissue albumin concentrations decreased between 210 and 630 days of age. Mesenteric loose connective tissue albumin-excluded volume fraction increased by 80% over this age range. The increase could not be accounted for by dehydration alone, suggesting that the increase in excluded volume fraction for albumin is also due to changes in tissue glycosaminoglycans or collagen.

Age-related changes in perimicrovascular protein distribution.

The diffusion hypothesis for physiological aging proposes that an increase in interstitial matrix fiber-to-gel ratio causes a decrease in nutrient diffusion to the cells. This hypothesis predicts a decrease in interstitial matrix protein with age. The objective was to test this hypothesis by determining age-related changes in plasma protein distribution in perimicrovascular and distal regions of rat mesentery interstitial matrix. Rats that were 77, 140, 210, 315, 455, and 630 days old were anesthetized with pentobarbital sodium, and a mesenteric loop was exteriorized. Intravital video microspectrophotometry was performed using wavelengths of 280, 320, and 700 nm. Perimicrovascular protein concentrations from the protein absorbance images were used to obtain the histogram, mean, and skewness of the proximal and distal protein concentration distributions. An exponential gradient model was also used to obtain the proximal and distal protein concentrations and gradient decay constants. Proximal protein concentration increased from 77- to 140-day-old rat and then decreased gradually through 210-, 315-, 455-, and 630-day-old rats. Distal concentration decreased gradually from 140- to 630-day-old rats. There was an increase in positive skewness of the proximal protein distributions from 140- through 630-day-old rats. We found an age-related decrease in perimicrovascular protein and propose that this is due to a decrease in protein permeability with age. The results support the diffusion theory of aging.

Age-related changes in rat interstitial matrix hydration and serum proteins.

Measurements of effects of age-related changes on loose connective tissue protein concentrations and water are lacking. Tissue hydration is an important determinant of tissue protein diffusion coefficients and hydraulic conductivity. Sprague-Dawley rats (ages 11, 30, 57, and 89 wk) were anesthetized. Blood and mesenteric tissue samples were taken. Tissue water content was determined by microgravimetric technique. Protein content was determined by electrophoresis. Tissue hydration decreased 18% between 11 and 89 wk with one-half the fall occurring between 57 and 89 wk. Tissue albumin concentration decreased 37% between 11 and 89 wk. Serum albumin increased 22% between 11 and 30 wk and then decreased by 37% to 1.4 g/dl in 89-wk rats. Serum total protein concentration increased by 13% between 11 and 89 wk, whereas tissue total protein concentration decreased 30% with age to 1.9 g/dl at 89 wk. The results suggest that age-related tissue dehydration is due, in part, to a shift in the colloid osmotic pressure components of Starling equilibrium favoring fluid reabsorption.

Changes in perimicrovascular protein spatial distribution due to superfusate.

OBJECTIVE: To determine superfusate-induced changes in the distribution of plasma proteins in the perimicrovascular interstitial matrix. METHODS: Rats were anesthetized with sodium pentobarbital and a mesenteric loop was exteriorized. Intravital video microspectrophotometry was performed using wavelengths of 280, 320, and 700 nm. The images were analyzed to give protein and collagen spatial distributions in vascular regions of rat mesenteric tissue. Perimicrovascular protein concentrations were fitted to an exponential decay model ci + cr exp (-x/k), where ci is distal protein concentration, ci + cr is the protein concentration proximal to the vessel, x is the distance from the vessel wall, and k is the decay constant indicating protein gradient slope. RESULTS: Before superfusion with 0.5-ml normal saline, ci = 1.45 +/- 0.13 g/dl, ci + cr = 4.56 +/- 0.23 g/dl. After the first superfusion, ci decreased (p < 0.01) to 0.53 +/- 0.06 g/dl; following a second superfusion, cr decreased to 0.4 +/- 0.03 g/dl; an additional final superfusion caused a further decrease to 0.33 +/- 0.02 g/dl. ci + cr also decreased significantly during repeated superfusions to 2.92 +/- 0.15, 2.35 +/- 0.25, and 2.1 +/- 0.12 g/dl, respectively. CONCLUSIONS: Superfusion produced changes in perivascular and distal interstitial matrix protein distribution. Protein concentration proximal to the microvessel remained higher than distal concentrations. This could be due to increased gel concentrations inhibiting protein mobility.

Changes in rat mesentery interstitial matrix due to superfusate.

Animal preparations for microscopy often require a superfusate solution to cover surgically exposed tissue. There are few, if any, data concerning the effects of this solution on extravascular protein concentration and hydration. The effect of superfusion on mesenteric tissue in anesthetized male Sprague-Dawley rats was studied. Tissue samples were taken from nonsuperfused and superfused tissue and analyzed for hydration, albumin, and transferrin content. The mesenteric tissue interstitial matrix was rapidly altered by normal saline superfusate. After superfusion, there was a decrease (P < 0.01) in tissue albumin concentration from 1.17 +/- 0.27 to 0.10 +/- 0.08 g/dl (n = 9). Tissue hydration increased from 4.98 +/- 0.8 micrograms water/microgram dry wt in controls to 7.38 +/- 1.2 micrograms water/micrograms dry wt after superfusion. When a range of superfusate albumin concentrations was used (0, 1, 2, and 3 g/dl), tissue albumin concentration changed 0.59 +/- 0.09 g/dl for each gram per deciliter change in superfusate concentration (P < 0.0001). The large changes in interstitial matrix protein content and hydration suggest that superfusate solution effects need to be considered in microvascular protein transport experiments.