REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

EFFECTS OF SERUM ON MEMBRANE TRANSPORT : II. Serum and the Stimulation of Adenosine Transport, a Possible Mechanism

When monolayers of freshly obtained rabbit lung macrophages are exposed to the nucleoside analogue, showdomycin (sho), adenosine transport, measured over a 45 s interval, is irreversibly inhibited. Low doses of the drug or short periods of exposure, however, do not result in decreased transport, while higher concentrations or longer exposures result in exponential decline. The initial lag is not due to a long reaction time of sho with the transport carrier or to nonspecific sites absorbing the drug. Previously it was shown that preincubation of monolayers with normal rabbit serum (NRS) results in increased adenosine transport. When monolayers are first exposed to sho so as to inhibit transport to varying degrees and then incubated with NRS, transport is increased over the inhibited level. Several experiments make it unlikely that serum removes the drug from the cell surface in a nonspecific fashion. Moreover, serum given before, during, or after sho alters the dose response curve so that no shoulder is seen. One way to explain these results makes use of target theory: the adenosine transport system could be comprised mainly of "coupled" or "clustered" sites of which only one is active at any time as well as "hidden" sites which are inactive. When a site in a group is irreversibly inactivated by sho, another in the group becomes activated. Serum might activate or uncouple all sites and also cause the appearance of hidden sites, which previously neither transported nor bound sho.     

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III₂ + IV (S₁) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.     

Knockdown of lncRNA MEG3 inhibits viability,migration, and invasion and promotes apoptosis by sponging miR‐127 in osteosarcoma cell

Osteosarcoma (OS) is one of the most common bone malignancies and occurs almost exclusively in children and adolescents. This study aimed to explore the role of lncRNA maternally expressed gene 3 (MEG3) in OS cells growth and metastasis, and to uncover the possible underlying mechanism. In this study, the expressions of MEG3 in five OS cell lines (MG63, OS‐732, SaOS, G292, and 143B) and in a human osteoblast cell line hFOB1.19 were measured by qRT‐PCR analysis. The expressions of MEG3, miR‐127, and ZEB1 in OS‐732 cells were overexpressed or suppressed by transfection. Cell viability, migration, invasion, and apoptosis were then assessed. The results showed that MEG3 was highly expressed in OS cell lines when compared to hFOB1.19 cell. MEG3 silence significantly suppressed OS‐732 cells growth and metastasis, as evidenced by the decreases in cell viability, migration, invasion, and increase in apoptotic cell rate. MEG3 acted as an endogenous sponge by binding to miR‐127. More interestingly, MEG3 silence could not suppress OS‐732 cells growth and metastasis when miR‐127 was knocked down. ZEB1 was a target gene of miR‐127, and miR‐127 overexpression‐induced impairments in cell growth and metastasis were attenuated when ZEB1 was overexpressed. Moreover,miR‐127 suppression activated JNK and Wnt signaling pathways, while these activations were recovered by ZEB1 silence. To conclude, our findings suggest that lncRNA MEG3 promoted OS cells growth and metastasis in vitro through sponging miR‐127. This study provides the evidence that MEG3 may be a potential therapeutic target for OS.     

miR‐185 affected the EMT,cell viability,and proliferation via DNMT1/MEG3 pathway in TGF‐β1‐induced renal fibrosis

Kidney fibrosis is usually the final manifestation of a wide variety of renal diseases. Recent years, research reported that long non‐coding RNAs (lncRNAs) played important roles in a variety of human diseases. However, the role and underlying mechanisms of lncRNAs in kidney fibrosis were complicated and largely unclear. In our study, we constructed the cell model of renal fibrosis in HK2 cells using transforming growth factor β1 (TGF‐β1) and found that lncRNA maternally expressed gene 3 (MEG3) was downregulated in TGF‐β1‐induced renal fibrosis. We then found that overexpressed MEG3 inhibited the TGF‐β1‐induced promotion of epithelial–mesenchymal transition, cell viability, and proliferation. Furthermore, we demonstrated that DNA methyltransferases 1 (DNMT1) regulated the MEG3 expression by altering the CpGs methylation level of MEG3 promoter in TGF‐β1‐induced renal fibrosis. In addition, we further revealed that miR‐185 could regulate the DNMT1 expression and thus, modulate the MEG3 in TGF‐β1‐induced renal fibrosis. Ultimately, our study illustrated that the modulation of the miR‐185/ DNMT1/ MEG3 pathway exerted important roles in TGF‐β1‐induced renal fibrosis. In summary, our finding displayed a novel regulatory mechanism for TGF‐β1‐induced renal fibrosis, which provided a new potential therapeutic target for renal fibrosis.     

LYSINE METABOLISM IN THE RAT BRAIN: BLOOD—BRAIN BARRIER TRANSPORT, FORMATION OF PIPECOLIC ACID AND HUMAN HYPERPIPECOLATEMIA

Through the use of intravenous pulse injection of L-[U-¹⁴C] lysine, the blood-brain barrier transport of L-lysine was studied. The uptake of L-lysine plus metabolites in the brain remained essentially unchanged at approx 0.002–0.005 nmol/g in the low dose (3μg per kg body weight) injection, and 20–40 nmol/g in the high dose (30 mg/kg) injection throughout the time intervals of up to 60 min. The uptake of L-lysine plus metabolites in the heart, however, decreased substantially from 0.03 to 0.003 nmol/g in the low dose injection and from 320 to 62 nmol/g in the high dose injection. The plasma to heart uptake ratio only decreased slightly through the 60 min period: from 6 to 2 in either the low or high dose L-lysine injection. The plasma to brain uptake ratio, however, decreased rapidly from a high of 62 to a low of about 4 in either the low or high dose injection throughout the 60-min time course. Study of labeled L-pipecolate formation in the plasma and individual organs indicates that this compound was formed only in the brain to a significant level within 0.5 min of ¹⁴C-L-lysine intravenous pulse injection. Labeled pipecolate was recovered from heart, liver, kidney and plasma in significant quantities only at 2 min or later after pulse-injection. It is concluded that the blood-brain barrier of L-lysine in the rat is not particularly strong and that the rat brain may be primarily responsible for L-pipecolate synthesis from L-lysine. The possible etiology of human hyperpipecolatemia is also discussed in light of the current findings.     

Length‐weight relationships for 32 fish species in the Grijalva River Basin,México

Length‐weight relationships were estimated for 32 species belonging to 11 fish families from the Grijalva River, México. For 24 of the species, the length‐weight relationships are published for the first time.     

Analysis of segregation and aneuploidy in two reciprocal translocation carriers, t(3;9)(q26.2;q32) and t(3;9)(p25;q32), by triple-color fluorescence in situ hybridization

Meiotic segregation patterns of chromosomes 3 and 9 were analyzed in sperm of two translocation carriers (t(3;9)(q26.2;q32) and t(3;9)(p25;q32)) by triple-color fluorescent in situ hybridization (FISH) with a telomeric DNA probe in addition to two centromeric probes. The frequencies of each sperm product resulting from alternate or adjacent I, adjacent II and 3:1 segregation in a t(3;9)(q26.2;q32) translocation carrier were 88.35%, 5.44% and 5.94%, respectively. On the other hand, the frequencies of each sperm product in a t(3;9)(p25;q32) translocation carrier were 89.23%, 6.02% and 4.48%, respectively. Of all the sperm products, the frequency of normal or chromosomally balanced sperm in a t(3;9)(q26.2;q32) and a t(3;9)(p25;q32) were 52.49% and 47.25%, respectively. The frequencies of each sperm product resulting from various segregations were different between both carriers and significantly deviated from the expected frequencies. Additional dual-color and triple-color FISH were performed to analyze aneuploidy rates for chromosomes 12, 17, 18, X and Y in order to detect any interchromosomal effect; no evidence of an interchromosomal effect was found.