Wall morphogenesis in diatoms: Deposition of silica by cytoplasmic vesicles

Summary Several TEM and SEM techniques were applied to examine developing structures in valves of the centric diatomThalassiosira eccentrica (Ehrenb.) Cleve after cytokinesis. It was possible to confirm that in each stage of the silicification process there is a distinction between a growing zone with a loose assemblage of silica spheres and a compacting zone in an older phase of development. The spherical structure of the silica in the growing zone results from the addition of silica by small cytoplasmic vesicles of about 300 to 400 Å in diameter. The vesicle membrane fuses with the silicalemma and the vesicle content is released into the silica-deposition vesicle. The origin of these vesicles, named STV, is still unknown.     

Cucurbit phloem serpins are graft-transmissible and appear to be resistant to turnover in the sieve element-companion cell complex

Serpins are unique inhibitors of serine proteases that are located in various plant tissues and organs. An orthologue of the pumpkin (Cucurbita maxima) phloem serpin CmPS-1 was amplified from cucumber (Cucumis sativus) RNA by RT-PCR, cloned, and designated as CsPS-1 (GenBank accession no. AJ866989). Alternative amino acid sequences in the reactive centre loop suggest distinct inhibitory specificity between CmPS-1 and CsPS-1. A difference in the electrophoretic mobility of these serpins was used in heterografts to establish that serpins are phloem-mobile. Immuno light microscopy revealed that the phloem serpins are localized exclusively to sieve elements (SE), while the phloem filament protein CmPP1, used as a reference, is localized to both SEs and companion cells (CCs). Similar to CmPS-1, CsPS-1 accumulates over time in phloem exudates, indicating that serpins differ from other phloem-mobile proteins whose concentrations appear to be stable in phloem exudates. These differences could reflect alternative mechanisms regulating protein turnover and/or inaccessibility of protein degradation. The functionality of the pore/plasmodesma units connecting SEs and CCs was tested with graft-transmitted CmPP1 as a transport marker. The occurrence of CmPP1 in the CCs of the Cucumis graft partner shows that translocated 88 kDa phloem filament protein monomers can symplasmically exit the SE and accumulate in the CC. By contrast, serial sections probed with the serpin antibody demonstrate that the 43 kDa serpin does not enter CCs. Collectively, these data indicate that CCs play a decisive role in homeostasis of exudate proteins; proteins not accessing the CCs accumulate in SEs and display a time-dependent increase in concentration.     

Tissue specification and intracellular distribution of actin isoforms inVicia faba L.

Summary Two dimensional gel electrophoresis of total cell protein extracts from not expanded, and primary leaves, petioles, and roots ofVicia faba resulted in four actin isoforms at 43 kDa with pI values from 5.9 to 6.05. In contrast to root extracts, in all leaf extracts an additional immunoreactive polypeptide with a molecular mass of 51 kDa and pI 5.75 was detected. This polypeptide was present in high amounts in protein extracts of purified chloroplasts, whereas no actin isoform at 43 kDa could be demonstrated. Compared to the tissue extracts, two actin isoforms at 43 kDa with pI values of 5.9 and 6.0 were enriched, when purified plasma membranes and the membranous fraction of vacuoles were analysed. In contrast, the soluble protein fraction of the plasma membrane preparation contained only two isoactins with pI values of 5.95 and 6.05 and a molecular mass of 43 kDa. These results indicate, that the four actin isoforms at 43 kDa detected in all examined tissues ofV. faba fulfill different functions at specific intracellular compartments, for example, the anchorage of actin microfilaments to membranes.Abbreviations BSA bovine serum albumin - BCIP 5-bromo-4-chloro-3-indolyl phosphate - DDM n-decyl -D-maltopyranoside - EDTA ethylenediamine-tetraacetic acid - HG n-hexyl -D-glucopyranoside - IEF isoelectrical focusing - MES morpholinoethanesulfonic acid - 2-ME 2 mercaptoethanol - NBT nitro blue tetrazolium - pCMB p-chloromercuribenzoic acid - PVP polyvinylpyrrolidone - Tris tris (hydroxymethyl) aminomethane     

Assessment of tissue damaging effects of mixed micellar absorption enhancers on the intestinal mucosa of common carp (Cyprinus carpio), African catfish (Clarias gariepinus) and rainbow trout (Oncorhynchus mykiss) as a consequence of enhanced intestinal absorption of sGnRH-a

Non-ionic surfactant–polyoxyethylene sorbitan monooleate (Tween-80) and oleic acid are food and pharmaceutical ingredients for oral and parenteral delivery and generally regarded as safe (GRAS). They have the potential as an intestinal absorption enhancer for the development of oral drug delivery systems. However, their safety in terms of mucosal integrity has yet to be evaluated. Therefore, the purpose of the present work was to study the tissue damaging effects of Tween-80, oleic acid and of their mixed micellar formulation (oleic acid + Tween-80). This was investigated at 2 h and 24 h after rectal delivery and compared with the topical effect of polyoxyethylene 9 lauryl ether (Polydocanol). The same experiment was carried out on three species with distinct feeding habits: the common carp, Cyprinus carpio (L.); the African catfish, Clarias gariepinus (Burchell); and the rainbow trout, Oncorhynchus mykiss (Walb.). Based on the findings of this study it was concluded that Tween-80 (4%), or its mixed micelle with oleic acid (0.6%) can be considered as a safe formulation, inducing only a moderate alteration of the intestinal mucosa, comparable to the effect of an isotonic saline. By contrast, in the three species, the same dose of Polydocanol, or of its mixed micelle with oleic acid, induced severe tissue damage of the intestinal mucosa, still present after 24 h. Mixed micelles of oleic acid with Tween-80 were also demonstrated as increasing the intestinal absorption of the salmon gonadotropin releasing hormone analogue (sGnRH-a) in catfish, C. gariepinus , and consequently stimulating GtH II secretion. This effect was compared with the action of other drugs considered as intestinal absorption enhancers.     

On the way to a quality control of the essential oil of fennel by means of Raman spectroscopy

Essential oils are one of the most valuable natural products. The price of special essential oils that can be purchased on the market strongly depends on the quality of the product. The quality, which depends on the quantitative and qualitative variation of different monoterpenes, varies with respect of the origin and the harvesting period. This contribution reports on a Raman spectroscopic study on the essential oil occurring in fennel. Cross-sections of fennel seed were investigated by use of Raman spectroscopy and Raman mapping to localize the essential oil and to analyze its chemical composition directly in the plant. Furthermore the practicability of a home-built mobile transportable Raman spectrometer to perform on-site measurements was successfully tested.     

Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

Background

The CXCR4 chemokine receptor regulates migration and homing of cancer cells to specific metastatic sites. Determination of the CXCR4 receptor status will provide predictive information for disease prognosis and possible therapeutic intervention. However, previous attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have produced predominant nuclear and occasional cytoplasmic staining but did not result in the identification of bona fide cell surface receptors.

Methodology/Principal Findings

In the present study, we extensively characterized the novel rabbit monoclonal anti-CXCR4 antibody (clone UMB-2) using transfected cells and tissues from CXCR4-deficient mice. Specificity of UMB-2 was demonstrated by cell surface staining of CXCR4-transfected cells; translocation of CXCR4 immunostaining after agonist exposure; detection of a broad band migrating at M _r 38,000–43,000 in Western blots of homogenates from CXCR4-expressing cells; selective detection of the receptor in tissues from CXCR4+/+ but not from CXCR4−/− mice; and abolition of tissue immunostaining by preadsorption of UMB-2 with its immunizing peptide. In formalin-fixed, paraffin-embedded human tumor tissues, UMB-2 yielded highly effective plasma membrane staining of a subpopulation of tumor cells, which were often heterogeneously distributed throughout the tumor. A comparative analysis of the mouse monoclonal antibody 12G5 and other frequently used commercially available antibodies revealed that none of these was able to detect CXCR4 under otherwise identical conditions.

Conclusions/Significance

Thus, the rabbit monoclonal antibody UMB-2 may prove of great value in the assessment of the CXCR4 receptor status in a variety of human tumors during routine histopathological examination.     

Effects of FGF2 and oxygen in the BMP4-driven differentiation of trophoblast from human embryonic stem cells

Human embryonic stem cells (hESC) differentiate into trophoblast when treated with BMP4. Here we studied the effects of either low (4% O₂, L) or atmospheric O₂ (20% O₂, A) in the presence and absence of FGF2 on H1 hESC cultured in the presence of BMP4. Differentiation progressed from the periphery toward the center of colonies. It occurred most quickly in the absence of FGF2 and under A and was slowest in the presence of FGF2 and under L. Chorionic gonadotropin (CG) production required A, while FGF2 suppressed progesterone synthesis under both A and L. FGF2 was then omitted while we examined the trophoblast markers SSEA-1 and cytokeratin-7 and-8, whose expression also progressed inward from the periphery of colonies and occurred more rapidly under A than under L. By day 5, most cells outside central islands of Oct4-positive cells were positive for these antigens under both conditions and many also expressed HLA-G, a marker of extravillous cytotrophoblast. Under A, but not L, CG and CGβ became prominent in GATA2-positive, peripherally located, multinucleated cells. In conclusion, BMP4 induced conversion of hESC exclusively toward trophoblast; FGF2 slowed differentiation, while O₂ accelerated this process and promoted syncytiotrophoblast formation.     

Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens

Pseudomonas fluorescens is able to grow on R-benzoin as the sole carbon and energy source because it harbours the enzyme benzaldehyde lyase that cleaves the acyloin linkage using thiamine diphosphate (ThDP) as a cofactor. In the reverse reaction, this lyase catalyses the carboligation of two aldehydes with high substrate and stereospecificity. The enzyme structure was determined by X-ray diffraction at 2.6 A resolution. A structure-based comparison with other proteins showed that benzaldehyde lyase belongs to a group of closely related ThDP-dependent enzymes. The ThDP cofactors of these enzymes are fixed at their two ends in separate domains, suspending a comparatively mobile thiazolium ring between them. While the residues binding the two ends of ThDP are well conserved, the lining of the active centre pocket around the thiazolium moiety varies greatly within the group. Accounting for the known reaction chemistry, the natural substrate R-benzoin was modelled unambiguously into the active centre of the reported benzaldehyde lyase. Due to its substrate spectrum and stereospecificity, the enzyme extends the synthetic potential for carboligations appreciably.