Purification, structural characterization, cloning and immunocytochemical localization of chemoreception proteins from Schistocerca gregaria.

Soluble low-molecular-mass protein isoforms were purified from chemosensory organs (antennae, tarsi and labrum) of the desert locust Schistocerca gregaria. Five genes encoding proteins of this group were amplified by PCR from cDNAs of tarsi and sequenced. Their expression products are polypeptide chains of 109 amino acids showing 40-50% sequence identity with putative olfactory proteins from Drosophila melanogaster and Cactoblastis cactorum. Direct structural investigation on isoforms purified from chemosensory organs revealed the presence in the expression products of two of the genes cloned. Two additional protein isoforms were detected and their molecular structure exhaustively characterized. MS analysis of all isoforms demonstrated that the four cysteine residues conserved in the polypeptide chain were involved in disulfide bridges (Cys29-Cys38 and Cys57-Cys60) and indicated the absence of any additional post-translational modifications. Immunocytochemistry experiments, performed with rabbit antiserum raised against the protein isoform mixture, showed selective labelling of the outer lymph in contact sensilla of tarsi, maxillary palps and antennae. Other types of sensilla were not labelled, nor were the cuticle and dendrites of the sensory cells. No binding of radioactively labelled glucose or bicarbonate was detected, in disagreement with the hypothesis that this class of proteins is involved in the CO2-sensing cascade. Our experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera.     

Evaluation of the growth rate of MCF-7 breast cancer multicellular spheroids using three mathematical models

Abstract. Growth data on 60 multicellular spheroids of MCF-7 human breast cancer cells were fitted, on an individual basis, by the Gompertz, Bertalanffy and logistic equations. MCF-7 spheroids, initiated and grown in medium containing oestrogens, exhibited a growth rate that decreased continuously as spheroid size increased. Plots of spheroid volume v. time generated sigmoid curves that showed an early portion with an approximately exponential volume increase; a middle region or retardation phase characterized by a continuously decreasing growth rate; and, finally, a late segment or plateau phase approaching zero growth rate, that permitted an estimate of the maximum spheroid size (V_max). Growth curves generated by MCF-7 spheroids under different experimental conditions (hormones, drugs and radiation exposures) can be compared after normalization. Linearized forms of the fitted Gompertz curves provided a convenient way to express differences in growth rate.     

Affinities of Nutty and Green-smelling Pyrazines and Thiazoles to Odorant-binding Proteins, in Relation with their Lipophilicity

Thirty-two green- and/or nutty-smelling compounds, most of thempyrazine and thiazole derivatives, were tested in ligand bindingassays with purified 19 kDa bovine and 22 kDa porcine olfactivebinding proteins (OBPs). Unlike the nutty ones, all the greenodorants were found to be good ligands for both proteins. Lipophilicityappears as an interesting discriminating physicochemical parameter,predictive of the affinity for OBPs and of the related odourprofile. Chem. Senses 20: 601–608, 1995.     

Expression of a Lipocalin in Human Nasal Mucosa

A 19 kDa soluble protein was purified from human nasal mucus. Its N-terminal amino-acid sequence appeared to be identical to that of a lipocalin synthesised both in lachrymal glands and in von Ebner's glands (VEG) of circumvallate papillae. In order to verify whether this protein was synthesised in the nasal cavity or was the result of tear contamination, we adopted an immunohistochemical approach. Polyclonal antibodies, raised against a primate VEG protein, were used on sections of human nasal mucosa obtained from surgery. The results clearly indicate that the protein is synthesised in sero-mucous glands underlying the respiratory ciliated epithelium. Although ligand-binding experiments with some odorant molecules have given negative results, we cannot exclude a role of odorant solubiliser and carrier for this protein.     

Identification of a rhodopsin photoreceptor in Euglena gracilis.

Visual pigments are a class of receptor proteins that absorb light and trigger sensory signals. Retinal-containing proteins are used in nature as photoreceptors mainly in animals vision. Mammalian rhodopsin is the best studied example of a light sensor which couples photon absorption to a cascade of biochemical reactions amplifying the input signal. A surprising discovery was to find rhodopsin also in Archaebacteria and in unicellular eukaryotes. On the basis of absorption microspectroscopic measurements and of inhibition experiments on pigment biosynthetic pathways, we have recently suggested that a rhodopsin could be the functional receptor of the visual process in Euglena gracilis, a flagellate which can use light directly to promote photosynthetic reactions, or as an incident flux of information to adjust its swimming orientation. We here report purification and identification of all-trans-retinal by column chromatography, HPLC and GC-MS in E. gracilis; these findings indicate with absolute certainty that rhodopsin is the photoreceptor molecule of this microorganism.     

Isolation of rat trachea interstitial fluid and demonstration of local cytokine production in lipopolysaccharide-induced systemic inflammation.

Access to interstitial fluid from trachea is important for understanding tracheal microcirculation and pathophysiology. We tested whether a centrifugation method could be applied to isolate this fluid in rats by exposing excised trachea to G forces up to 609 g. The ratio between the concentration of the equilibrated extracellular tracer 51Cr-labeled EDTA in fluid isolated at 239 g and plasma averaged 0.94 +/- 0.03 (n = 14), suggesting that contamination from the intracellular fluid phase was negligible. The protein pattern of the isolated fluid resembled plasma closely and had a protein concentration 83% of that in plasma. The colloid osmotic pressure in the centrifugate in controls (n = 5) was 18.8 +/- 0.6 mmHg with a corresponding pressure in plasma of 22 +/- 1.5 mmHg, whereas after overhydration (n = 5) these pressures fell to 9.8 +/- 0.4 and 11.9 +/- 0.4 mmHg, respectively. We measured inflammatory cytokine concentration in serum, interstitial fluid, and bronchoalveolar lavage fluid in LPS-induced inflammation. In control animals, low levels of IL-1 beta, IL-6, and TNF-alpha in serum, trachea interstitial fluid, and bronchoalveolar lavage fluid were detected. LPS resulted in a significantly higher concentration in IL-1 beta and IL-6 in interstitial fluid than in serum, showing a local production. To conclude, we have shown that interstitial fluid can be isolated from trachea by centrifugation and that trachea interstitial fluid has a high protein concentration and colloid osmotic pressure relative to plasma. Trachea interstitial fluid may also reflect lower airways and thus be of importance for understanding, e.g., inflammatory-induced airway obstruction.     

The structure of the gas bladder of the spotted gar,Lepisosteus oculatus

We report here on the macroscopic, light microscopic, and electron microscopic structure of the gas bladder (GB) of the spotted gar, Lepisosteus oculatus. The GB opens into the pharynx, dorsal to the opening of the oesophagus, through a longitudinal slit bordered by two glottal ridges. Caudal to the ridges, the GB is an elongated sac divided into a central duct and right and left lobes. The lobes are formed by a cranio‐caudal sequence of large air spaces that open into the central duct. The structure of the GB is that of a membranous sac supported by a system of septa arising from the walls of a central duct. The septa contain variable amounts of striated and smooth muscle might function to maintain the bladder shape and in providing contractile capabilities. The presence of muscle cells, nerves, and neuroepithelial cells in the wall of the GB strongly suggests that GB function is tightly regulated. The central duct and the apical surface of the thickest septa are covered by mucociliated epithelium. Most of the rest of the inner bladder surface is covered by a respiratory epithelium which contains goblet cells and a single type of pneumocyte. These two cell types produce surfactant. The respiratory barrier contains thick areas with fibrillar material and cell prolongations, and thin areas that only contain basement membrane material between the capillary wall and the respiratory epithelium. Lungs and GBs share many anatomical and histological features. There appears to be no clear criterion for structural distinction between these two types of respiratory organs. J. Morphol. 276:90–101, 2015. © 2014 Wiley Periodicals, Inc.     

Vergleichend experimentelle Untersuchungen über den Stoffwechsel des isolierten lebenden Kiemengewebes von Regenbogenforelle (Trutta iridea W. Gibb.) und Döbel (Squalius cephalus L.)

Ohne ZusammenfassungHerrn Professor Dr. B. Rensch und Herrn Oberregierungsrat Dr. H. W. Denzer sei für die Unterstützung der Arbeit herzlichst gedankt.     

CHARACTERIZATION AND DEVELOPMENTAL CHANGES OF UDP-GALACTOSE-CERAMIDE GALACTOSYL TRANSFERASE IN A RAT CNS AXOLEMMA-ENRICHED FRACTION. DIFFERENCES AND SIMILARITIES OF THE ENZYME ASSOCIATED WITH THE MICROSOMAL AND MYELIN FRACTIONS

Abstract— The properties of rat CNS UDP-galactose-ceramidc galactosyltransferase in an axolemma-enriched fraction (AXL), microsomes, and myelin simultaneously isolated with the AXL was characterized using a newly developed assay system. The microsomal enzyme utilized either magnesium or manganese equally well as the divalent cation at 3.3 m m , while both the myelin and AXL enzyme preferred manganese over magnesium at this concentration. The microsomal enzyme was more stable to heat inactivation than the myelin or AXL enzyme. The AXL galactosyltransferase had the highest specific activity at 15 days (8-fold higher than that of the microsomes) and dramatically decreased in specific activity with development. The developmental profile of the myelin enzyme paralleled that of the AXL although the absolute specific activity was lower than that of AXL. In contrast, the specific activity of microsomal enzyme was quite low at the earliest age then sharply increased to 25 days and gradually decreased with further development. The specific activity of the enzyme in AXL isolated from Quaking mouse was dramatically decreased (about 5% of control levels) whereas both whole homogenate and microsomal specific activity were decreased to 35% of control levels. These data indicate that AXL and myelin contain a galactosyltransferase with properties which are unique relative to those of the microsomal fraction. The possible functional significance of these findings with respect to myelination is discussed.