Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3)

The functional regulation of integrins is a major determinant of cell adhesion, migration and tissue maintenance. The binding of cytoskeletal proteins to various sites of integrin cytoplasmic domains is a key mechanism of this functional regulation. Expression of recombinant integrin alpha(IIb)beta(3) and alpha(M)beta(2) lacking the GFFKR-region in CHO cells results in constitutively activated integrins. In contrast, CHO cells stably expressing either a GFFKR-deleted alpha(V(del))beta(3) or a FF to AA-substituted alpha(V(AA))beta(3) do not reveal a constitutively activated integrin. Adhesion to immobilized fibrinogen is strongly impaired in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells, whereas it is not impaired in alpha(IIb)beta(3) and alpha(M)beta(2), both lacking the GFFKR-region. In a parallel plate flow chamber assay, alpha(V)beta(3)-expressing cells adhere firmly to fibrinogen and spread even at shear rates of 15 to 20 dyn/cm(2), whereas alpha(V(del))beta(3) or alpha(V(AA))beta(3) cells are detached at 15 dyn/cm(2). Actin stress fiber formation and focal adhesion plaques containing alpha(V)beta(3) are observed in alpha(V)beta(3) cells but not in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells. As an additional manifestation of impaired outside-in signaling, phosphorylation of pp125(FAK) was reduced in these cells. In summary, we report that the GFFKR-region of the alpha(V)-cytoplasmic domain and in particular two phenylalanines are essential for integrin alpha(V)beta(3) function, especially for outside-in signaling. Our results suggest that the two beta(3)-integrins alpha(IIb)beta(3) and alpha(V)beta(3) are differentially regulated via their GFFKR-region.     

Heterogeneity in rabbit liver cytochrome P-450 LM2 observed by cation exchange HPLC: Partial biochemical characterization of the two major LM2 subfractions

Cytochrome P450 LM2 (CYPIIB4) from phenobarbital-induced rabbit liver microsomes, purified to only one band in SDS-PAGE, was further resolved in five peaks by cation exchange HPLC. The two major peaks were partially characterized. Both of them have the amino terminal sequence Met-Glu and the same Cys content. They exhibited the same spectral absorption maximum and similar binding constants for 1-benzylimidazole and imidazole. However, binding of benzphetamine was different. One subfraction presented a Michaelis-Menten type binding curve, but the other presents a non-typical one with an additional high affinity binding site. These subfractions of cytochrome P450 LM2 slightly differed in their catalytic activities with benzyloxy- and pentoxyresorufin substrates. On the contrary, no heterogeneity was observed for P450 LM4.     

Morphological aspects of interactions between microparticles and mammalian cells: intestinal uptake and onward movement

Uptake of ingested microparticles into small intestinal tissues and on to secondary organs has moved from being an anecdotal phenomenon to a recognised and quantifiable process, which is relevant to risk assessment of accidental exposure, treatment of multi-organ dysfunction syndrome and therapeutic uses of encapsulated drug or vaccine delivery. This review puts in context with the literature the findings of a morphological study of microparticle uptake, using two approaches.The first is a rat in vivo in situ model, appropriate to a study rooted in the exposure of human populations to microparticles. Latex microspheres 2 μm in diameter are the principal particle type used, although others are also investigated. Most data are based on microscopy, but analysis of macerated bulk tissue is also useful. Uptake occurs at early time points after a single dose and is shown to take place almost entirely at villous rather than Peyer's patch sites: however, multiple feeding and therefore a longer time-span produces a higher proportion of particles associated with Peyer's patches, albeit for very small total uptake at those later time points. Uptake is less affected by species, fasting and immunological competence than by age and reproductive status.The second approach uses in vitro methods to confirm the role of intercellular junctions in particle uptake. Particle-associated tight junction opening, in a Caco-2 monolayer, is reflected in changes in transepithelial resistance and particle uptake across the epithelial monolayer: Tight junction opening and particle uptake are both increased further by external irradiation, ethanol and sub-epithelial macrophages, but reduced by exposure to ice. An M cell model has looser tight junctions than Caco-2 cells, but a similar level of particle uptake. These results, along with the changes seen in junctional proteins after particle addition, confirm the role of tight junctions in uptake but suggest that adhering junctions are also important.     

毛竹叶片衰老过程的叶重量、叶面积及元素内吸收率的动态

对福建永春毛竹(Phyllostachyspubescens Mazel ex H.de Lehaie)叶片衰老过程的叶重量、叶面积及元素内吸收率的动态进行了研究,并对元素内吸收率RE1(以元素的干重含量为计算单位,mg/g)、RE2(以单位叶片的元素含量为计算单位,mg/leaf)以及RE3(以单位叶面积的元素含量为计算单位,mg/cm2)进行了比较.叶片衰老过程中,平均叶重量、叶面积及比叶重分别下降了19.55%、15.16%和5.07%.叶重量与叶面积下降百分率的季节变化趋势一致,说明毛竹叶片存在一定的重量与面积比率.在不同的元素内吸收率比较中,N和K的元素内吸收率均为正,Ca均为负,表明叶片衰老过程中N和K的元素含量从衰老叶片中转移至植株的其他部位,而Ca在老叶中累积.N、P、K、Ca和Mg5种元素平均的元素内吸收率高低顺序均为RE2＞RE3＞RE1,反映出以元素的干重含量为计算单位和以单位叶面积的元素含量为计算单位的元素内吸收率偏低.     

Observations of spore morphology of some Pottiaceae Schimp. species (Bryophyta) in Turkey

The spores of Syntrichia ruralis (Hedw.) F. Weber and D. Mohr., S. princeps (De Not.) Mitt., S. subulata (Hedw.) F.Weber and D.Mohr var. subulata, S. subulata (Hedw.) F.Weber and D.Mohr var. angustata (Schimp.) J.J. Amann and S. subulata (Hedw.) F.Weber and D.Mohr var. graeffii Warnst. were studied by light and scanning electron microscopy for the first time. The apertural region consists of a leptoma in all spores. All taxa of the family are uniform in their spore morphology. The spores of the five taxa are of granuloid type. The spore wall of the Pottiaceae family includes sclerine (the distinction between exine and perine may be difficult to define) and intine. The taxonomy of the genus Syntrichia is discussed on the basis of its spore morphology.     

Cryopreservation of organs by vitrification: perspectives and recent advances

The cryopreservation of organs became an active area of research in the 1950s as a result of the rediscovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949. Over the ensuing four decades of research in this area, the advantages of vitrification, or ice-free cryopreservation, have become apparent. To date, experimental attempts to apply vitrification methods to vascularized whole organs have been confined almost entirely to the rabbit kidney. Using techniques available as of 1997, it was possible to vitrify blood vessels and smaller systems with reasonable success, but not whole organs. Beginning in 1998, a series of novel advances involving the control of cryoprotectant toxicity, nucleation, crystal growth, and chilling injury began to provide the tools needed to achieve success. Based on these new findings, we were first able to show that an 8.4M solution (VMP) designed to prevent chilling injury at -22 degrees C was entirely non-toxic to rabbit kidneys when perfused at -3 degrees C and permitted perfusion-cooling to -22 degrees C with only mild additional damage. We next investigated the ability of the kidney to tolerate a 9.3M solution known as M22, which does not devitrify when warmed from below -150 degrees C at 1 degrees C/min. When M22 was added and removed at -22 degrees C, it was sometimes [corrected] fatal, but when it was perfused for 25min at -22 degrees C and washed out simultaneously with warming, postoperative renal function recovered fully. When kidneys loaded with M22 at -22 degrees C were further cooled to an average intrarenal temperature of about -45 degrees C (about halfway through the putative temperature zone of increasing vulnerability to chilling injury), all kidneys supported life after transplantation and returned creatinine values to baseline, though after a higher transient creatinine peak. However, medullary, papillary, and pelvic biopsies taken from kidneys perfused with M22 for 25min at -22 degrees C were found to devitrify when vitrified and rewarmed at 20 degrees C/min in a differential scanning calorimeter. It remains to be determined whether this devitrification is seriously damaging and whether it can be suppressed by improving cryoprotectant distribution to more weakly perfused regions of the kidney or by rewarming at higher rates. In conclusion, although the goal of organ vitrification remains elusive, the prospects for success have never been more promising.     

Annealing of normal and mutant wheat starches. LM, SEM, DSC, and SAXS studies

Structure and thermodynamic properties of native and annealed wheat starches with different amylose content (from 1.5% to 39.5%) have been studied by high-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray diffraction (SAXS), light (LM), and scanning electron microscopy (SEM). Starch morphology, the values of the melting cooperative unit, the thickness of crystalline lamellae and the size of amylopectin clusters as well as thermodynamic parameters characterizing surface of the face side in starch crystals were determined. Some suppositions based on different physical approaches are used for a discussion of the results concerning structural reorganization of starches on different levels of macromolecular organization.     

The branched mitochondrial respiratory chain from Debaryomyces hansenii: Components and supramolecular organization

The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (_MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV₂, I–IV, III₂–IV₄, V₂, I–III₂, I–III₂–IV, I–III₂–IV₂, I–III₂–IV₃ and I–III₂–IV₄. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.     

Expression of ogu cytoplasmic male sterility in cybrids of Brassica napus

Summary A light and electron microscopic investigation revealed that ogu cytoplasmic male sterility (CMS) in cybrids of Brassica napus is primarily a deficiency of the tapetum and clearly time and site specific. Three patterns of ogu CMS were found, and specific conclusions drawn. First, the partially male fertile cybrid 23 was highly variable. It sometimes produced heterogeneous stamens with an endothecium formed exclusively around the fertile locules, thus delineating each microsporangium as a functional unit. The second type, including cybrids 27, 58 and 85, on the contrary, was stable and completely male sterile. In the four locules of normal length, microspores were observed to die at the vacuolate polarized stage while the tapetum disappeared prematurely through excessive vacuolization by the end of meiosis followed by a rapid autolysis during the tetrad or early free microspore stage. The subepidermal layer of the locule wall failed to form characteristic thickenings. The male-sterile stamens were completely indehiscent. At the time of anthesis they contained only collapsed empty exines adhering to each other. These cybrids, 27, 58 and 85, were closest to the ogu CMS trait of radish and seemed to be the best suited for further use in plant breeding. The third pattern was found in cybrids 77 and 118, which besides showing abortion of the microsporangia also showed a feminization of the stamens. We suggest that this feminization might be due to an alloplasmic situation associating Brassica napus nuclear genes with the mitochondrial DNA of radish.     

Origin and evolution of the unusual leaf epidermis of Caryodaphnopsis (Lauraceae)

We studied leaf epidermal anatomy of Caryodaphnopsis Airy Shaw, a genus disjunct between tropical Asia and tropical America, using light microscope and scanning electron microscope. We sampled 10 species of Caryodaphnopsis and 52 species of other Lauraceous genera. Our observations suggest that this genus possesses a unique lower leaf epidermis. Compared with other leaves of Lauraceae, this genus has an additional layer covering the lower leaf epidermis and the stomatal apparatus. The additional layer is either closed or poriferous/reticulate. The outer periclinal walls of the lower leaf epidermis protrude outside forming hollow domes or columns, and the distal endings of these domes or columns are expanded and fused, which forms the additional covering layer. Three different types are recognized in the genus: (1) the middle portion of the protuberances is not contracted and the distal endings are free or adnate to each other, with only limited space between the two layers; (2) the middle portion is slightly contracted in the outer part, the distal endings are fused, with more space between the two layers; (3) the middle portion is conspicuously contracted and elongated into columns, the distal endings are fused and it is roomy between the two layers. The structure of the leaf lower epidermis is reconstructed and illustrated for the first time. This unusual leaf lower epidermis of Caryodaphnopsis is derived in Lauraceae and is an autapomorphic character. Outward protrusions of the outer periclinal walls forming papillate protuberances surrounding the stomatal apparatus are also found in a few other genera including Neocinnamomum, a genus closely related to Caryodaphnopsis, but the distal endings of these protrusions are not expanded and connected. We hypothesize that the periclinal wall of the lower leaf epidermis has been gradually modified in Lauraceae, from a smooth pattern in most genera, to papillate pattern (e.g. Neocinnamomum), and to the double layered lower leaf epidermis in Caryodaphnopsis. The origin and evolution of this unique lower epidermis might have been related to the climatic cooling and aridification since the late Eocene.