A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas.

AIMS: To develop a sensitive real-time PCR-based protocol for the detection of Xanthomonas campestris pathovars from Brassica seed. METHODS AND RESULTS: A 5' nuclease real-time PCR assay was developed to screen Brassica spp. seed for the presence of X. campestris pathovars that cause black rot. The assay amplifies a 78-bp segment of the X. campestris hrpF gene and a 100-bp segment of the Brassica spp. 18S-25S internal transcribed spacer region. The Brassica spp. target provides an internal control for the amplification process to prevent false negatives that may arise from inhibitors that are often present in extracts from plant material. Whilst the primers were compatible with SYBR Green I assays, the use of fluorescently labelled probes in a 5' nuclease assay afforded greatest sensitivity and specificity. Seed batches carrying one artificially infected seed among 10,000 were readily detected using the assay. The multiplex real-time PCR assay permitted the rapid detection of pathogenic strains of X. campestris from bacterial colonies, Brassica seed and plants. CONCLUSIONS: Strains of X. campestris pathogenic to brassicas were readily detected from seed via a multiplex 5' nuclease real-time PCR assay. The real-time assay offers an improvement in sensitivity and a reduced turn-around time over the conventional multiplex PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR can be used to rapidly screen Brassica spp. seed batches for the presence of X. campestris pathovars. This assay provides a means for growers and the seed industry to be aware of the black rot status of their planting material, so that they may more effectively employ disease control measures or seed disinfection.

Regulation of caveolin and caveolae by cholesterol in MDCK cells.

We have examined the expression of caveolin in MDCK cells under conditions that vary cellular cholesterol concentration. Caveolin mRNA levels dropped to one-sixth of control levels after treatment with simvastatin, an inhibitor of cholesterol synthesis, or beta-trimethyl cyclodextrin (CD), a cholesterol sequestering drug. Both simvastatin and CD treatment decreased total cellular cholesterol levels to about 50% of control values. The potent activator of the sterol regulatory element, 25-hydroxycholesterol, showed no direct regulation of caveolin mRNA levels. Caveolin protein concentration was also decreased to 50% of control values in cholesterol-depleted cells, giving rise to a severe attenuation of caveolin expression detected by indirect immunofluorescence labeling. Quantitative electron microscopy showed a total loss of morphologically recognizable invaginated caveolae after these cholesterol depletion treatments. When the number of invaginated caveolae per cell was expressed as a function of the cellular cholesterol content, a threshold phenomenon was observed, suggesting that caveolae only form when the steady state cellular cholesterol is above 50% of control values. These findings indicate that caveolins, and caveolae, may play an important part in cellular cholesterol homeostasis.

L-arginine reduces human monocyte adhesion to vascular endothelium and endothelial expression of cell adhesion molecules.

BACKGROUND: Monocyte adhesion to endothelial cells is a key early event in atherogenesis. Because L-arginine has been shown to reduce atheroma and to decrease monocyte-endothelial cell adhesion in an animal model of atherosclerosis, we studied the effects of L-arginine on human monocyte adhesion to human endothelial cells and endothelial expression of cell adhesion molecules. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were grown to confluence, then incubated for 24 hours with arginine-deficient media to which was added saline (control), 100 or 1000 mumol/L L-arginine, 100 mumol/L D-arginine, 100 mumol/L NG-monomethyl-L-arginine (L-NMMA), or 100 mumol/L L-arginine with 100 mumol/L L-NMMA. Human monocytes obtained by elutriation were incubated for 1 hour with HUVECs, and adhesion was measured by light microscopy. Compared with control, monocyte adhesion was reduced by L-arginine (59 +/- 10%, P = .01) and increased by L-NMMA (123 +/- 20%, P = .01). Surface expression of cell adhesion molecules by HUVECs was assessed by an ELISA under the above conditions with and without stimulation with interleukin-1 beta. Expression of ICAM-1 was reduced with both concentrations of L-arginine compared with control in both the basal (43 +/- 12%, P < .01), and stimulated (46 +/- 15%, P < .01) states, which correlated with decreased levels of mRNA. Expression of VCAM-1 was reduced only in the stimulated state and only in the presence of 1000 mumol/L L-arginine (72 +/- 24%, P = .02). CONCLUSIONS: L-Arginine reduces human monocyte adhesion to endothelial cells and decreases expression of certain endothelial cell adhesion molecules.

Regulation of nonmuscle myosin light chain 3 gene expression in response to exogenous MLC3nm mRNA.

We have evaluated the regulation of the nonmuscle alkali myosin light chain isoform, MLC3nm, in mouse C2 myoblasts, in vitro. We altered the normal MLC mRNA profile of these cells, using stable transfection to introduce an exogenous pool of human MLC3nm mRNA. We used an isoform-specific, species-specific mouse MLC3nm cDNA probe to examine the response of the endogenous gene to the exogenous expression. At high cell density, expression of the endogenous mouse MLC3nm mRNA in transfectants is reduced to 50-70% of that in vector-transfected controls. These results suggest that a feedback mechanism operates in vitro, to regulate the size of the total MLC3nm mRNA pool. The down regulation of the mRNA for endogenous isoform is not detected at low cell density, suggesting that the mechanism may be density dependent and related to myoblast differentiation.

Differential regulation of tropomyosin isoform organization and gene expression in response to altered actin gene expression.

Phenotypically altered C2 myoblast cells, generated by the stable transfection of human nonmuscle actin genes (Schevzov, G., C. Lloyd, and P. Gunning. 1992. J. Cell Biol. 117:775-786), exhibit a differential pattern of tropomyosin cellular organization and isoform gene expression. The beta-actin transfectants displaying a threefold increase in the cell surface area, showed no significant changes in the pattern of organization of the high M(r) tropomyosin isoform, Tm 2, or the low M(r) tropomyosin isoform, Tm 5. In contrast, the gamma- and beta sm-actin gene transfectants, exhibiting a twofold decrease in the cell surface area, had an altered organization of Tm 2 but not Tm 5. In these actin transfectants, Tm 2 did not preferentially segregate into stress fiber-like structures and the intensity of staining was greatly diminished. Conversely, a well-defined stress fiber-like organization of Tm 5 was observed. The pattern of organization of these tropomyosin isoforms correlated with their expression such that a profound decrease in Tm 2 expression was observed both at the transcript and protein levels, whereas Tm 5 remained relatively unchanged. These results suggest that relative changes in nonmuscle actin gene expression can affect the organization and expression of tropomyosin in an isoform specific manner. Furthermore, this apparent direct link observed between actin and tropomyosin expression suggests that nonpharmacological signals originating in the cytoskeleton can regulate cytoarchitectural gene expression.

Differential regulation of the atrial isoforms of the myosin light chains during striated muscle development.

We have isolated a cDNA that encodes the human regulatory myosin light chain isoform predominant in adult atrial muscle. The cDNA contains an open reading frame of 175 amino acids and encodes a hydrophilic protein of a largely helical structure with two potential phosphorylation sites. The protein is different from any other regulatory myosin light chain so far described and is the product of a previously uncharacterized single copy gene. An isoform-specific probe was used to analyze the expression of this isoform in adult muscle and in cardiac and skeletal muscle development in vivo and in vitro. Parallel analysis of the corresponding human alkali myosin light chain (predominant in adult atrium) showed that both isoforms are expressed in early heart development, in both atrium and ventricle. Although the atrial alkali light chain is expressed throughout embryonic striated muscle development, the regulatory myosin light chain was not detected in skeletal myogenesis in vivo or in vitro. Thus the atrial isoforms are not universally or exclusively "paired" and can be independently regulated. We propose that the manner in which these particular isoforms fulfill the functional requirements of the muscle at different developmental times may have direct impact on their regulation.

Characterization of human myosin light chains 1sa and 3nm: implications for isoform evolution and function.

We have isolated a cDNA clone for the human slow-twitch muscle isoform myosin light-chain 1slow-a (MLC1sa) from a skeletal muscle library and for the human nonmuscle isoform myosin light-chain 3nonmuscle (MLC3nm) from a fibroblast library. The nucleotide sequence of both isoforms was determined, and isoform-specific probes were constructed. In addition, MLC1sa was subsequently isolated from the fibroblast library. MLC1sa and MLC3nm were found to be very closely related to each other and distant from all other myosin light-chain isoforms so far described. We concluded that MLC1sa arose by duplication of MLC3nm rather than from any other isoform. A comparison was made between all human myosin light chains described to date and a model proposed for the evolution of this multigene family. A comparison between human and chicken myosin light-chain isoforms showed that human isoforms are more similar to their chicken counterparts than to human MLC1sa. The expression of MLC1sa and MLC3nm was studied in humans, rabbits, mice, and rats. MLC1sa was detected at the onset of both human and murine myogenesis in vitro. With development, MLC1sa may be replaced by the other slow-twitch muscle isoform, 1sb, in slow-twitch skeletal muscle, but the proportion of MLC1sa to 1sb expression varies between different species. MLC1sa was detected in nonmuscle cells in humans, mice, and rats. MLC3nm was the major nonmuscle alkaline myosin light chain in all species tested, but its pattern of expression in nonmuscle tissues was not identical to that of beta- or gamma-actin. We have shown that in the human, as in the chicken, one exon is spliced out of the MLC3nm transcript in smooth muscle to give an alternative product. We concluded that all alkali myosin light-chain isoforms may be functionally different.