Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer

Summary The purified (H⁺ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H⁺-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H⁺)ATPase molecules were closely associated with liposomes, exhibiting a high H⁺-pumping activity (150,000% quenching· min^–1·mg^–1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H⁺)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H⁺)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles (inside-out orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H⁺)ATPase could also functionally insert into bilayer from PCPEPG or PCPEPI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H⁺)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H⁺)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation     

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation

Summary Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.     

Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritima.

Cakile maritima is a local oilseed halophyte exhibiting potential for secondary metabolite production. In the present study, plant growth, leaf polyphenol content and antioxidant activity were comparatively analyzed in two C. maritima Tunisian accessions (Jerba and Tabarka, respectively sampled from arid and humid bioclimatic stages) under salt constraint. Three-week-old plants were subjected to 0, 100, and 400 mM NaCl for 28 days under glasshouse conditions. A significant variability in salt response was found between both accessions: while Tabarka growth (shoot biomass, leaf expansion) was significantly restricted at 100 and 400 mM NaCl, compared to the control, Jerba growth increased at 100mM before declining at 400 mM NaCl. The better behaviour of Jerba salt-challenged plants, compared to those of Tabarka, may be related to their higher polyphenol content (1.56- and 1.3-fold the control, at 100 and 400 mM NaCl respectively) and antioxidant activity (smaller IC(50) values for both 1,1-diphenyl-2-picrylhydrazyl and superoxide scavenging), associated with lower leaf MDA accumulation (ca. -66% of the control at 100mM NaCl). Taken together, our findings suggest that halophytes may be interesting for production of antioxidant compounds, and that the accession-dependent capacity to induce antioxidative mechanisms in response to salt, may result in a corresponding variability for growth sustainability.     

Histological complexities of pancreatic lesions from transgenic mouse models are consistent with biological and morphological heterogeneity of human pancreatic cancer

Although pancreatic cancer is the fourth leading cause of cancer death, it has received much less attention compared to other malignancies. There are several transgenic animal models available for studies of pancreatic carcinogenesis, but most of them do not recapitulate, histologically, human pancreatic cancer. Here we review some detailed molecular complexity of human pancreatic cancer and their reflection in histomorphological complexities of pancreatic lesions developed in various transgenic mouse models with a special concern for studying the effects of chemotherapeutic and chemopreventive agents. These studies usually require a large number of animals that are at the same age and gender and should be either homozygote or heterozygote but not a mixture of both. Only single-transgene models can meet these special requirements, but many currently available models require a mouse to simultaneously bear several transgene alleles. Thus it is imperative to identify new gene promoters or enhancers that are specific for the ductal cells of the pancreas and are highly active in vivo so as to establish new single-transgene models that yield pancreatic ductal adenocarcinomas for chemotherapeutic and chemopreventive studies.     

Differential growth factor responses of epithelial cell cultures derived from normal human prostate,benign prostatic hyperplasia,and primary prostate carcinoma

Because of a lack of information of the optimum nutritional requirements, epithelial cells derived from normal human prostate and prostate tumors have been difficult to propagate in vitro, which hinders research in prostate carcinogenesis. In an effort to establish optimum nutritional conditions and differences in growth characteristics of normal human prostate (NP), benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCA), we have compared the effects of several growth factors on cell proliferation and elucidated growth properties of low passage epithelial cells derived from NP, BPH, and PCA of an African-American patient. Primary and low passage cultures were propagated in serum-free keratinocyte basal medium (KBM) supplemented with insulin (5 μg/ml), hydrocortisone (0.5 μg/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (BPE; 50 μg/ml), cholera toxin (10 ng/ml), and antibiotics. Almost all NP, BPH, and PCA cells were positive for cytokeratins and prostate-specific antigen (PSA). The NP, BPH, and PCA cells were essentially diploid and lacked mutations in c-K-ras and c-Ha-ras oncogenes, and p53 tumor suppressor gene. However, they exhibited progressively accelerating growth parameters. The population doubling times of NP, BPH and PCA were 51 hr, 37 hr, and 29 hr, respectively; their saturation densities were 2.9 × 10⁴/cm², 3.3 × 10⁴/cm², and 7.2 × 10⁴/cm², respectively. The NP and BPH cells required all of the growth factors in the medium, as deletion of any one of the above factors strongly inhibited their growth. The PCA cells, however, were independent of EGF and hydrocortisone. PC-3, an established human prostate cancer cell line, was independent of the growth factors tested. Fetal bovine serum (FBS) inhibited the growth of NP, BPH and PCA cells. In contrast, FBS stimulated the growth of the PC-3 cells in a concentration-dependent manner. These results indicate that in the absence of any apparent karyotype alterations and mutations in c-K-ras, c-Ha-ras and p53 genes, epithelial cells derived from NP, BPH, and PCA exhibit significant differences in their growth properties and responses to growth factors. These variations may represent early changes involved in prostate cancer, while gene mutations and cytogenetic alterations occur in advanced and/or metastatic tumors. © 1996 Wiley-Liss, Inc.     

14N and 15N imaging by SIMS microscopy in soybean leaves

The distribution of ¹⁵N and ¹⁴N compounds in cryofixed and resin embedded sections of soybean (Glycine max L) leaves was studied by SIMS microscopy. The results indicate that, with a mass resolution M/ΔM higher than 6000, images of the nitrogen distribution can be obtained from the mapping of the two secondary cluster ions ¹²C¹⁴N^? and ¹²C¹⁵N^?, in samples of both control and ¹⁵N-labeled leaves. The ionic images were clearly related to the histological structure of the organ, and allow the detection of ¹⁴N and ¹⁵N at the subcellular level. Furthermore, relative measurements of the ¹²C¹⁴N^? and ¹²C¹⁵N^? beams made possible the quantification of the ¹⁵N atom% in the various tissues of the leaf.     

Effect of sulphate deficiency on the plasma membrane polypeptide composition of Brassica napus

Highly purified oilseed rape (Brassica napus) root plasma membranefractions were prepared and their polypeptide patterns analysedby two-dimensional gel electrophoresis. Sulphur starvation enhancesthe sulphate uptake capacity of B. napus roots. The relativeabundance of several polypeptides increased significantly andspecifically after sulphur starvation. Several of them (37,38, 60, and 65 kDa), found in sulphur-starved plants, were moreabundant in a phase-partitioned membrane fraction treated withTriton X-100/KBr, indicating that they are intrinsic polypeptides.One polypeptide (47 kDa) was identified in the in vitro translationproducts of the roots mRNAs as specific for S-starved plants.It was also present among the intrinsic polypeptides specificfor —S plants. These plasma membrane polypeptides mightbe involved in sulphate uptake. Key words: Sulphate, sulphur-starvation, plasma membrane, polypeptides, root, transport