Immunocytochemical localization of epidermal growth factor in mouse submandibular gland.

The cellular and subcellular localization of epidermal growth factor in the submandibular glands of male and female adult mice was established by immunoperoxidase techniques. In light microscopic preparations epidermal growth factor was found exclusively in the granular convoluted tubules of the gland. The intensity of staining for epidermal growth factor varied from cell to cell, and some cells apparently were negative. The pattern of staining was similar in the glands of male and female mice; however, the granular convoluted tubules are androgen-responsive, and thus more extensive and composed of larger cells in males. In thin sections epidermal growth factor was most heavily concentrated in the secretion granules of the granular convoluted tubule cells. Within a given cell there was variation in intensity of staining of individual secretion granules, with some granules appearing minimally reactive or negative. The only other cell component with deposits of reaction product was the ribosomes.     

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.     

Immunocytochemical localization of renin in the submandibular gland of the mouse during postnatal development.

The localization of renin in the developing mouse submandibular gland was studied immunocytochemically using the unlabelled antibody-enzyme method of Sternberger ('74). Bouin-fixed submandibular glands of mice of both sexes were examined at 5-day-intervals from birth (day 0) to 50 days of age. At all stages studied, only granular convoluted tubule (GCT) cells stained immunocytochemically for renin; such cells were first seen in glands of 30-day-old males and of 30-day-old females. The size and number of renin-containing GCT cells increased rapidly in males, attaining adult status by 50 days of age. In females, differentiation of GCT cells immunoreactive for renin was slower and less regular than in males, and at 50 days of age the GCT segment had not yet reached adult conditions with respect to the distribution of renin. Renin appears in GCT cells at later ages than other GCT cell products (e.g., EGF and amylase), suggesting the existence of independent developmental control for the expression of various biologically active substances in the GCTs.     

Inflorescences vs leaves: a distinct modulation of carbon metabolism process during Botrytis infection

Plant growth and survival depends critically on photo assimilates. Pathogen infection leads to changes in carbohydrate metabolism of plants. In this study, we monitored changes in the carbohydrate metabolism in the grapevine inflorescence and leaves using Botrytis cinerea and Botrytis pseudo cinerea. Fluctuations in gas exchange were correlated with variations in chlorophyll a fluorescence. During infection, the inflorescences showed an increase in net photosynthesis (Pn) with a stomatal limitation. In leaves, photosynthesis decreased, with a non‐stomatal limitation. A decrease in the effective photosystem II (PSII) quantum yield (ΦPSII) was accompanied by an increase in photochemical quenching (qP) and non‐photochemical quenching (qN). The enhancement of qP and ΦPSII could explain the observed increase in Pn. In leaves, the significant decline in ΦPSII and qP, and increase in qN suggest that energy was mostly oriented toward heat dissipation instead of CO₂ fixation. The accumulation of glucose and sucrose in inflorescences and glucose and fructose in the leaves during infection indicate that the plant's carbon metabolism is differently regulated in these two organs. While a strong accumulation of starch was observed at 24 and 48 hours post‐inoculation (hpi) with both species of Botrytis in the inflorescences, a significant decrease with B. cinerea at 24 hpi and a significant increase with B. pseudo cinerea at 48 hpi were observed in the leaves. On the basis of these results, it can be said that during pathogen attack, the metabolism of grapevine inflorescence and leaf is modified suggesting distinct mechanisms modifying gas exchange, PSII activity and sugar contents in these two organs.     

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine

Non‐self‐recognition of microorganisms partly relies on the perception of microbe‐associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA‐regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long‐lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP‐non‐producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.     

Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes

The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.     

Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.     

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.