Immunofluorescent and immunochemical evidence for the expression of cytovillin in the microvilli of a wide range of cultured human cells

We have previously purified a Mr 75,000 protein, cytovillin, from cultured human choriocarcinoma cells (JEG-3) and shown that this protein was specifically confined to the microvillus membrane of these cells. I have now studied the expression and the subcellular distribution of cytovillin in eighteen normal and transformed human cell lines and strains by using immunoblotting and indirect immunofluorescence microscopy. In all cell types, cytovillin was highly enriched in cell surface protrusions. When cell types were ranked according to their staining intensity, choriocarcinoma was highest, then amniotic epithelial cells, other choriocarcinoma cells and tumor cells, and finally fibroblastoid cells. The latter only gave faint diffuse fluorescence on the plasma membrane and, occasionally, on the microvilli. However, detergent extracts of all cell types could be shown to contain cytovillin by the use of immunoblotting techniques. Metabolic pulse-chase labelling experiments with JEG-3 cells demonstrated synthesis of cytovillin as a single-chain polypeptide. No precursor forms or specific proteolytic cleavage products could be seen either by immunoblotting or immunoprecipitation. The protein was found to be very stable with a biologic half-life of about 25 hours. The pI determined by isoelectric focusing was 6.1. These results were consistent with cytovillin being an integral component of the microvilli and other surface extensions of all human cell types examined.     

Diurnal variations of medial basal and anterior hypothalamic thyroliberin [TRH] and serum thyrotropin [TSH] concentrations in male rats.

The diurnal variation of TRH concentrations in different parts of hypothalamus was studied in 80 male rats, which were killed in groups of 5 at 3 h intervals. The hypothalamus was dissected into three parts: I) medial basal hypothalamus (MBH), II) anterior hypothalamus, and III) dorsal hypothalamus. Anterior pituitary and serum TSH concentrations were also measured. TRH concentrations were higher in MBH than in the other parts of the hypothalamus: at night 300–450 pg/mg of wet weight of tissue. When the lights were turned on, MBH-TRH levels began to decrease, reaching a nadir of 210 pg/mg at 12 noon. After 15 h, MBH-TRH levels began to increase again. The changes in TRH levels in anterior hypothalamus were usually opposite to those in MBH (r = ?0.6185). Serum TSH levels were about 800 ng/ml during the day and were decreased to about one half of these levels when the lights were turned off. Serum TSH levels were positively correlated with anterior hypothalamic TRH levels (r = 0.6457) and inversely correlated with MBH-TRH levels (r = ?0.7747). Anterior pituitary TSH levels showed small but statistically insignificant variations. In conclusion, there were statistically interrelated diurnal rhythms in anterior hypothalamic and MBH-TRH levels and serum TSH concentrations.     

Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase I null mice

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)]2beta2, [alpha(II)]2beta2, and [alpha(III)]2beta2 tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.     

Effects of bovine colostrum supplementation on serum IGF-I, IgG, hormone, and saliva IgA during training

Mero, Antti, Heidi Miikkulainen, Jarmo Riski, RaimoPakkanen, Jouni Aalto, and Timo Takala. Effects of bovinecolostrum supplementation on serum IGF-I, IgG, hormone, and saliva IgAduring training. J. Appl. Physiol.83(4): 1144-1151, 1997.The purpose of this study was to examinethe effects of bovine colostrum supplementation (Bioenervi) on seruminsulin-like growth factor I (IGF-I), immunoglobulin G, hormone, andamino acid and saliva immunoglobulin A concentrations during a strengthand speed training period. Nine male sprinters and jumpersunderwent three randomized experimental training treatments of 8 daysseparated by 13 days. The only difference in the treatments was thedrink of 125 ml consumed per day. Posttraining increases were noticedfor serum IGF-I in the 25-ml Bioenervi treatment (125 ml contained 25 ml Bioenervi) and especially in the 125-ml Bioenervi treatment (125 mlcontained 125 ml Bioenervi) compared with the placebo (normal milkwhey) treatment (P < 0.05). The change in IGF-I concentration during the 8-day periods correlated positively with the change in insulin concentration during the sameperiods with 25-ml Bioenervi treatment(r = 0.68;P = 0.045) and with 125-ml Bioenervitreatment (r = 0.69;P = 0.038). Serum immunoglobulin G,hormone, and amino acid and saliva immunoglobulin A responses weresimilar during the three treatments. It appears that a bovine colostrumsupplement (Bioenervi) may increase serum IGF-I concentration inathletes during strength and speed training.

     

Interactions between extraradical ectomycorrhizal mycelia, microbes associated with the mycelia and growth rate of Norway spruce (Picea abies) clones

Despite their ecological relevance, field studies of the extraradical mycelia of ectomycorrhizal (ECM) fungi are rare. Here we examined in situ interactions between ECM mycelia and host vigour. Ectomycorrhizal mycelia were harvested with in-growth mesh bags buried under Norway spruce (Picea abies) clones planted in 1994 in a randomized block design. Mycelial biomass was determined and fungal species were identified by denaturing gradient gel electrophoresis (DGGE) and sequencing of the internal transcribed spacer 1 (ITS1) region. Microbial community structure in the mycelium was investigated by phospholipid fatty acid (PLFA) profiling. Compared to slow-growing spruce clones, fast-growing clones tended to support denser mycelia where the relative proportions of Atheliaceae fungi and PLFAs indicative of Gram-positive bacteria were higher. Ascomycetes and PLFAs representative of Gram-negative bacteria were more common with slow-growing clones. In general, the ECM mycelial community was similar to the ECM root-tip community. Growth rate of the hosts, the ECM mycelial community and the microbes associated with the mycelium were related, suggesting multitrophic interactions between trees, fungi and bacteria.     

Temporal Variation in Seed Predation by Insects in a Population of Syagrus romanzoffiana (Arecaceae) in Santa Catarina Island, SC, Brazil

Insect seed predation may vary depending on seed production. The present study considers the hypothesis that the rates of seed predation tend to be smaller in years of higher fruit production. Thus, we monitored the production of fruits and predation of seeds of the palm Syagrus romanzoffiana over 2?years in the Atlantic Forest (Parque Municipal da Lagoa do Peri, Florianópolis, SC, Brazil), between July 2006 and June 2008. Plots of 0.25?m² were fitted under 20 mother plants and fruits were monthly collected for assessment of abundance and seed predation. There was variation in fruit production between the 2?years and among reproductive plants. Predation rates were high and occurred in the predispersal phase by the Curculionidae Revena rubiginosa Boheman, Anchylorhynchus aegrotus Fahraeus, and Anchylorhynchus variabilis Gyllenhal. Seed predation by these species of Anchylorhynchus is first registered in the present study. In average, about 60% of the seeds monthly produced in the population tend to escape insect predation in year of high or low production, becoming available for recruitment. The predation rate was not related to the amount of fruits produced per reproductive plant. Also, different than expected, there was a positive relation between the rates of seed predation and the total of fruits produced monthly on the plots. Thus, no evidence for the satiation of insect seed predators was found in this study with S. romanzoffiana.     

The reactivity of ruthenium mono(bipyridine) carbonyl complexes in an alcoholic solution of alkali metal carbonates

Chlorine containing ruthenium bipyridine carbonyl compounds react readily in dilute alkaline solutions under a CO atmosphere affording a poorly soluble and air sensitive product that is suggested to have a polymeric nature. Various analysis methods (MS and TPD) were used in the characterisation of the product. The replacement of the axial chloride ligands in trans(Cl), cis(CO)[Ru(bpy)(CO)₂Cl₂] and [Ru(bpy)(CO)₂Cl]₂ is proposed to be the initial step in the polymerisation. The replacement of chlorides in methanolic solution was confirmed by isolating and characterising the dimeric intermediate [Ru(bpy)(CO)₂(COOCH₃)]₂.     

Cytovillin, a microvillar Mr 75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localization

Cytovillin is a microvillar cytoplasmic peripheral membrane protein, with prominent expression in vivo in placental syncytiotrophoblasts and certain human tumors. Cytovillin cDNA was cloned from a human placental lambda gt11 library using affinity purified antibodies. The identity of cytovillin cDNA clones was confirmed by expression of cytovillin in Escherichia coli and using antibodies raised against the expressed fusion protein in comparison with antibodies against cytovillin purified from cultured human choriocarcinoma cells. In these cells Northern blotting analysis identified a major 3.5-kilobase cytovillin mRNA. The cDNA encodes a protein of 575 amino acids corresponding to a molecular weight of 68,084. According to secondary structure prediction, cytovillin is a hydrophilic protein with an extensive internal alpha-helical region ending in a sequence of 7 consecutive prolines. The predicted alpha-helical region showed limited homology to alpha-helical regions of cytoskeletal proteins and certain other proteins, but no extensive homologies were found in the cytovillin cDNA or the deduced amino acid sequence to other registered DNA or protein sequences. Southern blot analysis of a DNA panel of human mouse somatic cell hybrids localized the cytovillin gene to the end of the long arm of chromosome 6 (6q22-q27). Our results show that cytovillin is representative of a novel class of microvillar proteins.