Vasoactive intestinal peptide increases intracellular cAMP and gonadotropin-alpha gene activity in JEG-3 syncytial trophoblasts. Constraints posed by desensitization.

Expression of the human chorionic gonadotropin (hCG)-alpha gene in placental trophoblasts is markedly stimulated by cAMP, a property preserved in a reporter plasmid containing its cAMP response elements (CREs) linked to the chloramphenicol acetyltransferase coding sequence (CRE alpha CAT). In search of a potential physiologic regulator of hCG gene expression via cAMP, we found that JEG-3 syncytial trophoblast cells have specific binding sites for vasoactive intestinal peptide (VIP) with dissociation constant of 1 nM. VIP maximally increased the transient expression of CRE alpha CAT and the expression of endogenous hCG-alpha mRNA in JEG-3 cells by 4- and 9-fold, respectively. Exposure of JEG-3 cells to 30 nM VIP increased cAMP levels 60-fold after 10-30 min, but cAMP rapidly declined thereafter. As a consequence of this desensitization, the effect of VIP on stimulation of both CRE alpha CAT and endogenous hCG-alpha and hCG-beta mRNA levels more closely resembled that of forskolin or 8-br-cAMP at time points much less than 24 h. Moreover, transient exposure to 8-br-cAMP was much less effective than 24 h of continuous incubation on CRE alpha CAT activity. We conclude that VIP rapidly increases cAMP content and activates hCG-alpha gene expression in JEG-3 cells, but sustained elevations in cAMP are necessary for maximal accumulation of this CRE-regulated gene product.     

Triphasic effect of prostaglandins E1, E2 and F2α on the fluid transport of isolated gall-bladder of guinea-pigs

Prostaglandins (PGs) F_2α, E₁ and E₂ exerted a triphasic influence on the fluid transport of isolated guinea-pig gall-bladders, when applied to the serosal side. PGE₁ and PGE₂ produced these effects in lower concentrations than F_2α. Directly after PG addition to the serosal side a short stimulation of fluid transport to between 200 and 400% was observed. The stimulatory effect of PGs was most distinct in gall-bladders from female guinea-pigs, less pronounced in male and nearly absent in pregnant animals. Since PGs increased intraluminal hydrostatic pressure in gall-bladders by contraction of the smooth muscle, experiments were performed in which hydrostatic pressure was increased by different procedures. These included the addition of imidazole (10⁻² M), raising of K⁺ in the bathing solution and an increase in intraluminal pressure by addition of Ringer's solution into the lumen. All three procedures stimulated fluid reabsorption temporarily in the same way as PGs, hence increase of intraluminal pressure is thought to be the reason for the observed temporary stimulation of fluid transport. Direct evidence for this thesis was obtained when the gall-bladder was mounted as a flat sheet over a chamber; in this preparation no stimulation of fluid transport was obtained. The second phase of the PG influence was characterized by a concentration-related inhibition of fluid reabsorption followed by a significant but small reverse of fluid transport (secretion of fluid). When PGs were applied to the mucosal side, only an inhibition of fluid transport was observed, which was much weaker compared to the addition to the serosal side.     

Association between CSN3 and BCO2 gene polymorphisms and milk performance traits in the Czech Fleckvieh cattle breed

Daily milk, fat and protein yield and amount of somatic cells in cow milk are very important factors that influence milk performance traits. An association between polymorphisms in the kappa casein (CSN3) gene and milk production, composition and technical properties has been previously reported; however, this type of information is not available for the bovine β-carotene oxygenase 2 (BCO2) gene--the BCO2 gene has relationship with milk color and meat fat color, which is dependent on content of β-carotene. We analyzed these two genes and their relationship with milk performance traits (daily milk, fat and protein yield, somatic cell count, SCC) in one cattle population, Czech Fleckvieh (N = 152). All animals were milked twice a day and kept in the same environmental conditions. The Fleckvieh is a typical Czech cattle breed farming for milk and meat production. It is the most common breed in the Czech Republic. DNA was isolated from milk or from hairs. Genes were analyzed using PCR-RFLP, frequencies of alleles and genotypes were calculated and association analysis was performed using a GLM Procedure in SAS. Statistical analysis established that the CSN3 gene has no statistically significant influence on daily milk, fat and protein yield and SCC. Compared to other references this result can be explained by, e.g., small group of animals and different cattle breed. The BCO2 gene (genotypes AA and AG) shows a statistically significant relationship (P = 0.05) with daily milk, protein yield and SCC.     

Positive feedback favors invasion by a submersed freshwater plant

The submersed macrophyte Utricularia inflata has invaded lakes in northern New York State, thereby threatening native isoetids such as Eriocaulon aquaticum. Isoetids often dominate and modify softwater lakes due to their capacity to oxidize sediment and thus influence solute mobilization. Greenhouse experiments tested the hypotheses that U. inflata invasion could result in higher porewater iron (Fe) concentrations and greater ammonium (NH₄ ⁺) and Fe release from the sediment into the water column, and that this mobilization would stimulate further U. inflata growth. In the first experiment, three levels of U. inflata impact on E. aquaticum were imposed using sediment cores overlain by lake water: E. aquaticum alone, E. aquaticum with a cover of U. inflata, and bare sediment—the latter to simulate local extirpation of the isoetid by the invasive. After 16 weeks, sediment porewater NH₄ ⁺ and total dissolved Fe concentrations were significantly higher (P < 0.05) for the U. inflata and bare sediment treatments. Water column concentrations of these solutes were five-fold higher (P < 0.05) for the bare sediment treatment than E. aquaticum alone, indicating that isoetid extirpation by U. inflata can compromise water quality. A second experiment demonstrated that U. inflata grew faster over bare sediment than over sediment with E. aquaticum (P < 0.05), likely due to greater solute mobilization in the absence of E. aquaticum. Where U. inflata causes a decline of native isoetids in Adirondack Mountain lakes, changes to lake sediment and water chemistry can create a positive feedback loop further escalating the impact of this invasive species.     

Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells

The uptake of methotrexate by KB cells was observed to be dependent on time, temperature, and concentration of extracellular methotrexate. The Kd for methotrexate surface binding to KB cells was approximately 200 nM. Following exposure of KB cells to trace quantities of [3H]methotrexate for periods ranging from 6 min to 24 h, the cellular methotrexate was progressively formed into methotrexate polyglutamates and was bound to dihydrofolate reductase as well as to a particulate folate binding protein. To further study the mechanism of methotrexate uptake in KB cells, the N-hydroxysuccinimide ester of methotrexate was used to covalently label the surface of KB cells and to inhibit transport of methotrexate. The N-hydroxysuccinimide ester of methotrexate was bound to a species of protein with an apparent molecular weight of 160,000 in 1% (v/v) Triton X-100 that bound folic acid and was specifically precipitated by antiserum raised against the previously purified high-affinity folate binding protein (the folate receptor) from human KB cells. In addition, trypsin was utilized to remove surface-accessible covalently bound methotrexate. The amount of covalently bound methotrexate that could be released by trypsin initially decreased on incubation at 37 degrees C, suggesting that the methotrexate and binding protein were internalized. However, with time, trypsin could again release the covalently bound methotrexate, suggesting that the binding protein cycles from the external cell surface to the inside of the cell and out again.     

Immunohistochemical detection of nestin in pediatric brain tumors.

Nestin is an intermediate filament protein (IFP) expressed in undifferentiated cells during CNS development and in CNS tumors. Previous studies have arrived at different conclusions in terms of which types of CNS tumors express nestin. In this report we establish an immunohistochemical protocol using antigen retrieval, which significantly enhances staining with two polyclonal anti-nestin antisera, #130 and #4350. The staining pattern was identical for the two nestin antisera and very similar to that of vimentin, while glial fibrillary acidic protein (GFAP), immunoreactivity was absent from 9.5-week-old forebrain. The current study of 20 primary CNS tumors from pediatric patients included seven ependymomas, seven primitive neuroectodermal tumors (PNETs), five pilocytic astrocytomas, and one glioblastoma multiforme (GBM). All these tumors expressed nestin to various extents, in contrast to five brain metastases tested. Strong nestin immunoreactivity was found in malignant primary CNS tumors, whereas benign pilocytic astrocytomas showed low but consistent nestin expression. In all tumors nestin immunoreactivity was confined to the cytoplasm of tumor cells and was co-expressed with astrocyte markers vimentin, GFAP, and S-100. Vascular endothelial cells of all neoplasms also showed marked immunoreactivity for nestin and vimentin, whereas they were negative for GFAP and S-100. In conclusion, antiserum #4350 detected nestin in formalin-fixed, paraffin-embedded tissue sections by heat-induced antigen retrieval immunohistochemistry. Nestin was expressed in both highly malignant and low malignant gliomas, indicating the potential use of nestin as a diagnostic tumor marker in surgical pathology.     

Micro Flow-Through Thermocycler with Simple Meandering Channel with Symmetric Temperature Zones for Disposable PCR-Devices in Microscope Slide Format

Chip-based flow-through PCR implements the PCR as a continuous process for nucleic acid analytics. The sample is transported in a winding channel through temperature zones required for denaturation, annealing and extension. Main fields of application are the monitoring of continuous processes for rapid identification of contaminants and quality control as well as high throughput screening of cells or microorganisms. A modular arrangement with five heating zones for flow-through PCR is discussed and evaluated. The special heater arrangement allows the implementation of up to 40 cycles on the footprint of a microscope slide, which is placed on top ofa 5 zones heating plate. Liquid/liquid two phase flow of PCR reaction mixture and mineral oil have been applied to create a segmented flow process scheme. In that way, the developed system may provide flow-through PCR as a unit operation for the droplet based microfluidics platform. The single use of disposable devices is commonly preferred due to the sensitivity of the PCR process to contaminations. All-glass microfluidic chips and disposable chip devices, made from polycarbonate as a replication with identically geometry, have been fabricated and tested. For the first time, microchannel geometries with nearly circular profile developed by all-glass technology have been transferred to mass fabrication by injection compression molding. Both devices have been successfully applied for the detection of the tumor suppressor gene p53. Although product yield and selectivity of the amplification process do not depend on the chip material, a well defined, reliable segmented flow regime could only be realized in the all-glass chip.     

A high-confidence human plasma proteome reference set with estimated concentrations in PeptideAtlas

Human blood plasma can be obtained relatively noninvasively and contains proteins from most, if not all, tissues of the body. Therefore, an extensive, quantitative catalog of plasma proteins is an important starting point for the discovery of disease biomarkers. In 2005, we showed that different proteomics measurements using different sample preparation and analysis techniques identify significantly different sets of proteins, and that a comprehensive plasma proteome can be compiled only by combining data from many different experiments. Applying advanced computational methods developed for the analysis and integration of very large and diverse data sets generated by tandem MS measurements of tryptic peptides, we have now compiled a high-confidence human plasma proteome reference set with well over twice the identified proteins of previous high-confidence sets. It includes a hierarchy of protein identifications at different levels of redundancy following a clearly defined scheme, which we propose as a standard that can be applied to any proteomics data set to facilitate cross-proteome analyses. Further, to aid in development of blood-based diagnostics using techniques such as selected reaction monitoring, we provide a rough estimate of protein concentrations using spectral counting. We identified 20,433 distinct peptides, from which we inferred a highly nonredundant set of 1929 protein sequences at a false discovery rate of 1%. We have made this resource available via PeptideAtlas, a large, multiorganism, publicly accessible compendium of peptides identified in tandem MS experiments conducted by laboratories around the world.     

Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon.

Connective tissue polarity has remained an intractable enigma for over two decades. We present new data on optical second harmonic generation in native, wet, rat-tail tendon. Scanning second-harmonic microscopy has revealed, for the first time, the existence of a discrete network of fine, polar, filamentous or columnar, structures, and, also, the presence of strongly polar surface, or near-surface patches. The thickness of these features was probed via crossed-beam optical frequency summation and the polar material is estimated to occupy a few percent of the tendon volume. The three-dimensional spatial distribution of filaments was studied with the aid of small-angle second-harmonic scattering, and the filaments were found to permeate the tendon cross-section in an apparently random fashion. These latter measurements also revealed that essentially all polar filaments had the same directionality. Concomitant studies of the polar collagen fibrils that comprise the bulk of tendon were in full accord with prior electron microscope results that had demonstrated that the directionality of these fibrils varies up/down in a purely random fashion, and thus cannot yield a net macroscopic polarity. Quantitative analysis of the second-harmonic data yields the conclusion that the observed polar structures cannot be simply local regions containing some accidental net excess of similarly oriented fibrils. The analytical expressions used in the analysis of the data obtained for this complex tissue were supported by extensive, realistic computer simulations. The discovery that the polarity of rat-tail tendon, and possibly other forms of connective tissue, resides in discrete structures, some of which are located near the tendon surface, should permit the ready isolation of polar-rich material for further study by a variety of techniques.