Study on placental transmission of Pneumocystis carinii in mice using immunodeficient SCID mice as a new animal model.

Placental transmission of Pneumocystis carinii in mice was examined in 39 animals obtained by caesarean section from 17 pregnant SCID females experimentally infected with P. carinii. When examined with toluidine blue O, DAPI and immunofluorescent antibody stains, P. carinii was detected in the lungs of infected mothers but not in the lungs of caesarean section-derived neonates even after the neonates were treated with dexamethasone for 8 weeks. However, 13 neonates born to five infected females developed P. carinii pneumonia. These results indicate that P. carinii cannot be transmitted transplacentally in mice.     

Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids.

Based on the ability to complement the poor growth of an M13 phage derivative lacking the complementary strand origin, eleven single-strand initiation sequences (ssi) for DNA replication are identified in the F, R6K, R100 and ColE2 plasmids. Six of them were from F, two from near the gamma and alpha origins (ori) of R6K, two from the vicinity of the basic replicon of R100 and one from near the ori of ColE2. They can be classified into two groups based on the morphology of the plaques and the length of nucleotide (nt) sequences required for ssi activity; one group that gives rise to larger and clearer plaques and can be reduced to nearly 100 nt (seven out of eleven), and another that generates smaller and less clear plaques and requires more than 200 nt for full activity (four out of eleven). Sequence homology is detected among some members from both groups. The possible biological roles of the ssi are discussed.     

Cell type-specific expression of the human renin gene.

We have previously produced transgenic mice carrying the human renin gene, whose expression is regulated in a tissue-specific manner. In the present study, we further characterized expression of the transgene. Northern blot analysis showed that the human renin gene is expressed in the kidney but not in the liver of two lines of transgenic mice with 10 and 50 copies of the transgene, suggesting that the integrated copy number of the human renin gene does not influence the dominant-renal expression pattern. Immunohistochemical study using a monoclonal antibody specific for human renin demonstrated that expression of human renin in the transgenic mouse kidney is confined to the epithelioid juxtaglomerular cells. Transfection experiments indicated that the chloramphenicol acetyltransferase fusion gene containing the 3-kb upstream sequences of the renin gene is activated only in human epithelioid embryonic 293 cells derived from kidney but not in human HepG2 cells from liver. These findings suggest that transfer of the cloned renin gene into mice and in vitro cultured cell lines can give rise to cell type-specific expression.     

REPRODUCTIVE ISOLATION AND DISTINCT POPULATION STRUCTURES IN TWO TYPES OF THE FRESHWATER SHRIMP PALAEMON PAUCIDENS

Twenty local populations of the Japanese freshwater shrimp Palaemon paucidens were electrophoretically and morphologically surveyed. Based on the diagnostic distributions of some alleles at Gpi, Mpi, Mdh-1, and Mdh-2, these populations were largely classified into two types (A and B). The A type occurred in lakes, ponds, and rivers, while the B type was observed only in rivers. Average Nei's genetic distance (D) between the two types fell into the subspecies range . The coefficient of gene differentiation, G_ST, varied considerably between the two types. In 12 populations of the A type, with a G_ST value of 0.281, nine pond and lake populations showed a higher G_ST (0.246) than the three river populations (0.151). On the other hand, G_ST was 0.036 for the eight local populations of the B type. The lower rostrum tooth number had a mode of two in type A and three in type B. Type-A populations largely varied in the upper rostrum tooth number and egg size but type B did not. Under laboratory conditions, mating frequently occurred within each type, but not between types. Furthermore, no embryonic development was observed in the few cases of intertype mating. These results indicate that the A and B types had experienced cladogenic separation with pre- or postmating isolation, whereafter the A type, under geographic isolation, underwent genetic and phenotypic differentiation, while the B type, under extensive gene flow, did not undergo differentiation.     

Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli.

Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly.     

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.     

Effect of changes in thyroid state on metabolism of thyroxine by rat placenta

We studied the effect of the state of the thyroid on T4 monodeiodination in the rat placenta, and it was compared with those in the liver and kidney. The tissues, maternal serum, and amniotic fluid were obtained from pregnant rats. The tissues were homogenized in cold 50 mM Tris-HCl buffer, pH 7.5. The homogenate (1 mg protein) was incubated at 37 degrees C for 60 min with 1 microgram T4 in the presence of 5 mM DTT. The T3 and reverse T3 generated in the reaction mixture were extracted into cold ethanol and measured by RIAs. The conversion of T4 to reverse T3 in rat placenta was not significantly changed in MMI-induced hypothyroidism or T4 induced hyperthyroidism. On the other hand, conversion of T4 to T3 in the liver and kidney were changed in parallel with the thyroid state. The concentration of reverse T3 in the amniotic fluid was increased in accordance with the increase in the maternal serum T4 concentration. These results indicate that the placental T4 inner ring deiodination is not affected by the thyroid state, and that the change in the amniotic fluid reverse T3 concentration in this study is mainly dependent upon the change in maternal thyroid function.     

Novel Method of Lactic Acid Production by Electrodialysis Fermentation

In lactic acid fermentation by Lactobacillus delbrueckii, the produced lactic acid affected the lactic acid productivity. Therefore, for the purpose of alleviating this inhibitory effect, an electrodialysis fermentation method which can continuously remove produced lactic acid from the fermentation broth was applied to this fermentation process. As a result, the continuation of fermentation activity was obtained, and the productivity was three times higher than in non-pH-controlled fermentation. In electrodialysis fermentation, the amount of produced lactic acid was 82.2 g/liter, which was about 5.5 times greater than that produced in non-pH-controlled fermentation. It was concluded that these good results were obtained on account of alleviating the lactic acid inhibitory effect by electrodialysis fermentation. However, the fouling of anion-exchange membranes by cells was observed in electrodialysis fermentation.