A new experimental approach for the study of cardiopulmonary physiology during early development

In this report, an experimental approach and newly designed apparatus for liquid ventilation of preterm animals are described. Findings of age-related changes in cardiopulmonary function of this animal preparation are presented. Thirty-one lambs, 102-137 days gestation (term 147 +/- 3 days), were studied. The carotid artery, jugular vein, and trachea of the exteriorized fetus were cannulated under local anesthesia. Immediately after cesarean section delivery, ventilation commenced; warmed (39 degrees C) and oxygenated (PIO2 greater than 500 Torr) liquid fluorocarbon (RIMAR 101) was delivered to the lung by a mechanically assisted liquid ventilation system. Skeletal muscle paralysis, low-dose exogenous buffering, and thermal support were maintained during the 3-h experiment. Pulmonary gas exchange, acid-base status, and cardiopulmonary and metabolic function were assessed. By utilizing these techniques, effective arterial oxygenation, CO2 elimination, acid-base status, and cardiovascular stability were supported independent of gestational age. The results demonstrate a developmental increase in specific lung compliance and mean arterial pressure and decrease in heart rate and systemic O2 consumption per kilogram with advancing gestational age. These findings demonstrate that liquid ventilation negates the dependency of effective pulmonary gas exchange on surfactant development, thereby extending the limits of viability of the immature extrauterine lamb. As such this new experimental approach is useful for the study of physiological development over an age range previously limited to fetal animal preparations and, therefore, may provide insight regarding adaptation of the premature to the extrauterine environment.     

Neplanocin A. Actions on S-adenosylhomocysteine hydrolase and on hormone synthesis by GH4C1 cells

We have investigated the biochemical actions of Neplanocin A (Nepl A), a carbocyclic adenosine analog, on purified calf liver S-adenosylhomocysteine hydrolase and in the GH4C1 strain of functional rat pituitary cells. Addition of 1 mol of Nepl A/2 mol of S-adenosylhomocysteine hydrolase subunit led to rapid and complete inactivation. Concomitant with inactivation, half of the enzyme-bound NAD was reduced and adenine was released stoichiometrically from Nepl A. In GH4C1 cells Nepl A caused a dose-dependent rapid (within 5 min) and irreversible inactivation of S-adenosylhomocysteine hydrolase and concomitant increase in intracellular S-adenosylhomocysteine. In cells treated with Nepl A for 4-5 days, methylation of DNA cytosine was depressed approximately 50%, and the level of cytoplasmic prolactin mRNA was elevated 2-fold. While acute (30 min) release of prolactin from intracellular stores was unaffected, Nepl A acted in a dose- and time-dependent manner to increase the production of both prolactin and growth hormone, the two hormones synthesized and secreted by GH4C1 cells. The lowest effective dose was 0.12 microM, the concentration required to decrease S-adenosylhomocysteine hydrolase activity by 50%. By 4-7 days the production of both hormones in Nepl A-treated cells was increased 2-3 times above control. The action on hormone production persisted for at least 7 days after removal of Nepl A from the culture medium. We conclude that Nepl A inhibits S-adenosylhomocysteine hydrolase, raises cellular S-adenosylhomocysteine, decreases bulk DNA methylation, and increases hormone synthesis in GH4C1 cells.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Evidence for replication slippage in the evolution of Oenothera chloroplast DNA.

Evolutionary relationships of four plastid genomes (plastomes) from different Oenothera species have been assessed by sequence comparisons of two intergenic regions that separate the ribosomal protein genes rpl16, rpl14, and rps8. Sequence changes include base substitutions, the occurrence of a 29-base tandem duplication, and variation in the length of two poly-A stretches. Additions/deletions in chloroplast DNA may not be useful for evolutionary comparisons more distant than these, particularly if the sequences undergo divergence after the initial event, but the length mutations reported here allow a finer resolution of the phylogeny of the closely related Oenothera plastomes than would have been possible if only base substitutions had been considered. Comparisons with the orthogous sequence from tobacco chloroplast DNA indicate the direction of change at most of the sites. The results suggest that plastomes I and II are closely related to each other, as are plastomes III and IV. Replication slippage is proposed as a mechanism to explain the length mutations.     

Comparison of gas and liquid ventilation: clinical, physiological, and histological correlates.

To differentiate the effects of gas and liquid ventilation on cardiopulmonary function during early development, we compared the clinical, physiological, and histological profiles of gas- and liquid-ventilated preterm lambs (n = 16; 108-116 days gestation). Immediately after cesarean section delivery, ventilation commenced using gas delivered by a volume ventilator (n = 9) or liquid perfluorochemical (n = 7) delivered by a mechanically assisted liquid ventilation system. Pulmonary gas exchange, acid-base status, vital signs, and respiratory compliance were assessed during the 3-h protocol; sections of the lungs were obtained for histological analyses when the animals were killed. Six of nine gas-ventilated lambs expired from respiratory failure before 3 h, with the remaining animals experiencing severe respiratory insufficiency, pneumothoraces, and cardiovascular deterioration. Six of seven liquid-ventilated lambs survived with good gas exchange and cardiovascular stability and without fluorothorax; one experienced ventricular fibrillation before 1 h and expired despite pulmonary stability. Respiratory compliance was significantly greater in the liquid- than in the gas-ventilated lambs. Histological analyses of gas-ventilated lungs demonstrated nonhomogeneous lung expansion, with thick-walled gas exchange spaces containing proteinaceous exudate, hemorrhage, and hyaline membranes. In contrast, liquid-ventilated lungs appeared clear, with thin-walled and uniformly expanded gas exchange spaces that were free of hyaline membranes and luminal debris. Morphometric analyses demonstrated that surface area and gas exchange index were greater in the liquid- than in the gas-ventilated lambs. These results indicate that elimination of surface active forces by liquid ventilation during early development provides more effective gas exchange with less barotrauma compared with gas ventilation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pentobarbital anesthesia: Lack of effect on venous prostaglandins in dogs

Venous prostaglandins A, E, and F were determined by radioimmunoassay in 10 dogs before and one hour after administration of sodium pentobarbital (35 mg/Kg, iv). In the conscious state, PGA was 0.34 ± 0.04 ng/ml (mean ± SE), PGE 0.20 ± 0.01 ng/ml, and PGF 0.25 ± 0.03 ng/ml. During pentobarbital anesthesia, these levels were unchanged (p >0.05). Thus, pentobarbital anesthesia had no effect on peripheral venous prostaglandin levels.     

Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent

Taxol is used in chemotherapy regimens against breast and ovarian cancer. Treatment of tumor model cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. Our results demonstrate that in response to taxol, various cell types differentially utilize distinct apoptotic pathways. Using MCF7 breast carcinoma cells transfected with caspase-3 gene, we showed that taxol-induced apoptosis occurred in the absence of caspase-3 and caspase-9 activation. Similar results were obtained with ovarian SKOV3 carcinoma cells, expressing high level of endogenous caspase-3. In contrast, staurosporine-induced apoptosis in these cells was accompanied by proteolytic cleavage of pro-caspase-3 and induction of caspase-3 enzymatic activity. The effect of taxol appears to be cell type-specific, since taxol-induced apoptosis in leukemia U937 cells involved caspase-3 activation step. We conclude that a unique caspase-3 and caspase-9 independent pathway is elicited by taxol to induce apoptosis in human ovarian and breast cancinoma cells.