Fast sampling,rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Summary Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10–40 l) with an incubation medium (0.2–1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4/sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15–20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na⁺-d-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.     

The effects of isotonic contractions on the rate of fatigue development and the resting membrane potential in the sartorius muscle of the frog, Rana pipiens.

The goal of this study was to characterize how isotonic contractions affect the rate of fatigue development. Muscle bundles dissected from frog sartorius muscles were stimulated with 100-ms long train of pulses (0.5 ms, 6 V, 140 Hz). To measure the effect of the isotonic contractions, isometric tetanus were elicited at regular time intervals during the stimulation to fatigue. In general, isotonic contractions caused a faster decrease in tetanic force than isometric contractions. The difference in tetanic force between an isotonic and isometric fatigue increased gradually over a 20-min period to 7.9 and 13.5% at 0.04 and 0.1 trains/s (TPS), respectively. At 0.2, 0.5, and 1.0 TPS, the decrease in tetanic force was also faster during an isotonic fatigue, which resulted in an initial difference in tetanic force between the two types of fatigue. The difference did not exceed 18.5% and did not persist throughout the stimulation period; i.e., the difference disappeared before the end of the fatigue stimulation. The half-relaxation time was prolonged during fatigue development, and the prolongation was greater during an isotonic fatigue, except at 0.04 TPS. The increases in the half-relaxation time at 0.2, 0.5, and 1.0 TPS were followed by a decrease, and the decreases were especially pronounced during an isotonic fatigue at 0.5 and 1.0 TPS. The results showed for the first time that isotonic contractions cause a faster rate of fatigue development in frog sartorius muscles, and this effect depends on the frequency of stimulation.     

Mechanisms involved in parasitic castration: in vitro effects of the trematode Prosorhynchus squamatus on the gametogenesis and the nutrient storage metabolism of the marine bivalve mollusc Mytilus edulis

The mechanisms involved in the parasitic castration of the marine mussel Mytilus edulis by the trematode parasite Prosorhynchus squamatus Odhner, 1905, have been investigated in vitro with two bioassays employing dissociated host tissues. There is no conclusive evidence that P. squamatus affects the secretion of two host neuroendocrine factors, viz., gonial mitosis-stimulating factor and glycogen mobilization hormone, involved in the gametogenesis/nutrient storage cycles of the mussel. In contrast, extracts of P. squamatus sporocysts and cercariae significantly stimulated glycogen mobilization in host glycogen cells and strongly inhibited host gonial mitosis. A gonial mitosis-inhibiting factor (GMIF) was found in the hemolymph of parasitized mussels. The existence of an endogenous GMIF in mantle tissue of uninfected mussels has been demonstrated. This factor appeared to be secreted into the hemolymph during the period of sexual maturity. Whether the parasite acts directly on the host gonia, or by provoking the liberation of this endogenous GMIF, has yet to be ascertained. It would appear, however, that the parasite acts directly on host glycogen cells.     

Differential susceptibility to a trematode parasite among genotypes of the Mytilus edulis/galloprovincialis complex

We show that parasitism by the trematode Prosorhynchus squamatus in parental and introgressed Mytilus edulis/galloprovincialis (Bivalvia) mussels occurs in individuals with a predominantly M. edulis genome. This result suggests that the restricted specificity of P. squamatus is dependent on genetic factor(s) present in M. edulis. Because of its strong pathogenic effects (i.e. total castration and possible death), this parasite may be a source of intense selection against M. edulis genomes when they are present in a site. As a consequence, it may favour the geographic extension of the M. galloprovincialis genome. Previous studies have indicated that, in hybrid zones, recombinant genotypes are more susceptible to parasitic infections than either parental genotype. We demonstrate that this is not the case for the M. edulis/M. galloprovincialis system, and that the parental genotype alone determines susceptibility.     

Progress in outgrowth culture from rabbit tracheal explants: Balance between proliferation and maintenance of differentiated state in epithelial cells

Summary Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively. This work was supported by DRET and by CIFRE grant awarded to S. R.     

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-hexanoic derivatives of 5 alpha-dihydrotestosterone and testosterone.

The synthesis and characterization of 17 alpha-(6'-hexanoic acid) derivatives of 5 alpha-dihydrotestosterone and testosterone, useful as ligands for affinity chromatography purification or as precursors for affinity-labeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3 beta-hydroxy-, and 3 beta,5-dihydroxy-5 alpha-androstan-17-one precursors with the potassium derivative of 5-hexyn-1-ol led to the corresponding 17 alpha-(6'-hydroxyhex-1'-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17 alpha-(6'-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-17-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17 alpha-(5'-carboxypentyl)-5 alpha-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3 beta-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3 beta,5 alpha-dihydroxy precursor resulted in a 5 alpha-hydroxy-3-oxo intermediate, which was dehydrated to give 17 alpha-(5'-carboxypentyl)testosterone. The 17 alpha configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their 1H and 13C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17 alpha- or 17 beta-substituted steroid analogs as well as with 1H and 13C NMR reference data recorded in this work for 17 alpha-ethynyltestosterone, 17 alpha-ethynyl-19-nortestosterone, 17 alpha-ethyl-19-nortestosterone, 17 alpha-methyltestosterone, and 17 alpha-methyl-5 alpha-dihydrotestosterone.     

Pleiotropic action of parasites: How to be good for the host

Parasites reduce the reproductive output of their hosts, limit their growth, and sometimes even castrate or hill them. Under certain conditions however, a parasitized host may be better off than an uninfected one. Such 'nice' parasites have a 'pleiotropic' action on their hosts. Parasites can be pleiotropic either in space (in which case they have a beneficial effect on the host in one environment while being detrimental in another) or in time (the parasite is beneficial at one stage of the host's development and 'costly' at another stage). Such pleiotropic parasites may constitute the intermediate stage between parasitism and mutualism.     

Tg (9 HSA-MYC), a homozygous lethal insertion in the mouse

Transgenic mice generated with different DNA sequences were surveyed for possible homozygous mutant phenotypes. We found an embryonic lethal mutation in the transgenic mouse strain (MT-MYC12.4) containing the human c-myc gene. Embryos homozygous for the transgene die shortly after implantation. The strain MT-MYC12.4 carries approximately 50 tandem copies of the recombinant plasmid sequence. The 3 flanking sequence has been cloned and analyzed. It contains a unique sequence that has been conserved during evolution and maps to Chromosome (Chr) 9. This mutant has been designated Tg 9 (HSA-MYC).     

Effects of Physicochemical Factors on the Adhesion to Cellulose Avicel of the Ruminal Bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. succinogenes

Ruminococcus flavefaciens adhered instantly to cellulose, while Fibrobacter succinogenes had the highest percentage of adherent cells after about 25 min of contact between bacteria and cellulose. Adhesion of R. flavefaciens was unaffected by high concentrations of sugars (5%), temperature, pH, oxygen, metabolic inhibitors, and lack of Na⁺. In contrast, the attachment was affected by the removal of divalent cations (Mg²⁺ and Ca²⁺), the presence of cellulose derivatives (methylcellulose and hydroxyethylcellulose), and cystine. Adhesion of F. succinogenes was sensitive to low and high temperatures, high concentrations of glucose and cellobiose (5%), hydroxyethylcellulose (0.1%), redox potential, pH, lack of monovalent cations, and the presence of an inhibitor of membrane ATPases or lasalocid and monensin. Cells of F. succinogenes heated at 100°C no longer were adherent. On the other hand, adhesion was insensitive to the lack of divalent cations (Mg²⁺ and Ca²⁺), the presence of 2,4-dinitrophenol, tetrachlorosalicylanilide, or inhibitors of the electron transfer chains. Adhesion of F. succinogenes seems to be related to the metabolic functions of the cell. External proteins and/or cellulases themselves might play a part in the attachment process. Several mechanisms are probably involved in the adhesion of R. flavefaciens, the main one being the interaction between the large glycocalyx and the divalent cations Ca²⁺ and Mg²⁺. Hydrophobic bonds and enzymes may also be involved.     

Effects of light,phosphate, and oxygen on ethylene formation and conidiation in surface cultures ofpenicillium cyclopium westling

Penicillium cyclopium growing in a surface culture with 2-ketoglutarate or glutamate as a sole carbon source produced ethylene in two phases. The first peak of ethylene production (EP 1) was associated with aerial mycelium growth whereas the second peak of ethylene production (EP 2) occurred with formation and maturation of conidia. Conidiation was induced by blue light between 120 and 172 h after the culture was started and depended on the presence of a carbon source at the stage of conidiophore initiation. Exogenous phosphate content dropper rapidly before the onset of conidiation. The EP 2 was connected with conidiation via this drop. Addition of phosphate prior to the conidiophore initiation and during conidiation inhibited EP 2 without affecting conidiation, but conidia lacked a green pigment and their germination ability decreased by 905. Exogenous ethylene did not restore normal development. The EP 2 in asporogenic cultures was evoked by incubation in the dark and by phosphate removal. The EP 2 and conidiation were accompanied by an increased oxygen consumption. The EP 1 yield of ethylene depended only on biomass growth and was unaffected by any treatment mentioned above.