Small RNA-induced mRNA degradation achieved through both translation block and activated cleavage.

Small RNA (sRNA)-induced mRNA degradation occurs through binding of an sRNA to a target mRNA with the concomitant action of the RNA degradosome, which induces an endoribonuclease E (RNase E)-dependent cleavage and degradation of the targeted mRNA. Because many sRNAs bind at the ribosome-binding site (RBS), it is possible that the resulting translation block is sufficient to promote the rapid degradation of the targeted mRNA. Contrary to this mechanism, we report here that the pairing of the sRNA RyhB to the target mRNA sodB initiates mRNA degradation even in the absence of translation on the mRNA target. Remarkably, even though it pairs at the RBS, the sRNA RyhB induces mRNA cleavage in vivo at a distal site located >350 nucleotides (nt) downstream from the RBS, ruling out local cleavage near the pairing site. Both the RNA chaperone Hfq and the RNA degradosome are required for efficient cleavage at the distal site. Thus, beyond translation initiation block, sRNA-induced mRNA cleavage requires several unexpected steps, many of which are determined by structural features of the target mRNA.

Mechanism of insulin-stimulated clearance of plasma nonesterified fatty acids in humans.

Insulin increases plasma nonesterified fatty acid (NEFA) clearance in humans, but whether this is independent of change in plasma NEFA appearance is currently unknown. Nine nondiabetic men (age: 28+/-3 yr, body mass index: 27.2+/-1.7 kg/m2) underwent euglycemic clamps to maintain low (LINS) vs. high (HINS) physiological insulin levels for 6 h. An intravenous infusion of heparin+Intralipid (HI) was performed during 4 of the 6 h of the clamps (in the last 4 h at LINS and in the first 4 h at HINS), whereas saline infusion (SAL) was administered in the remaining 2 h to modulate plasma NEFA levels independently of plasma insulin levels. Four experimental conditions were obtained in each individual: LINS with saline (LINS/SAL) and with HI infusion (LINS/HI) and HINS with saline (HINS/SAL) and with HI infusion (HINS/HI). Plasma palmitate appearance during HINS/SAL was lower than during the three other experimental conditions (P<0.05). In contrast, plasma linoleate appearance, as expected, was increased by HI independently of insulin level (P<0.02). Plasma palmitate clearance during HINS/SAL was higher than LINS/SAL and LINS/HI (P<0.008), and this increase was blunted during HINS/HI. We observed a linear decrease in plasma palmitate clearance with increasing plasma NEFA appearance independent of insulin levels. Plasma NEFA levels increased exponentially with increase in plasma NEFA appearance. We conclude that insulin stimulates plasma NEFA clearance by reducing the endogenous appearance rate of NEFA. The relationship between plasma NEFA level and appearance rate is nonlinear.

The effect of insulin on the intracellular distribution of 14(R,S)-[18F]Fluoro-6-thia-heptadecanoic acid in rats.

PURPOSE: The aim of this study was to determine the effect of hyperinsulinemia on myocardial and hepatic distribution and metabolism of 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA). PROCEDURES: Mitochondrial retention and intracellular lipid incorporation of [18F]FTHA were compared to that of [14C]-2-bromopalmitate or [14C]palmitate during hyperinsulinemic clamp vs. saline infusion in male Wistar rats. RESULTS: Mitochondrial 18F activity was increased in the heart (1.7 +/- 0.4 vs. 0.5 +/- 0.1% ID/g, P < 0.05), whereas it was reduced in the liver (1.1 +/- 0.3 vs. 1.8 +/- 0.4% ID/g, P < 0.05) during insulin vs. saline infusion, respectively. Mitochondrial [14C]-2-bromopalmitate activity was affected by insulin in a similar way in both tissues. The fractional esterification of [18F]FTHA into triglycerides was impaired compared to [14C]palmitate in both tissues, and [18F]FTHA was insensitive to the shift of esterification of fatty acids into complex lipids in response to insulin. CONCLUSIONS: [18F]FTHA is sensitive to insulin-induced modifications of free fatty acid oxidative metabolism in rats but is insensitive to changes in nonoxidative fatty acid metabolism.

New methods characterizing avalanche behavior to determine powder flow.

PURPOSE: To characterize the avalanche behavior of different powders and to compare the results of the strange-attractor and novel characterization approaches. METHODS: The following nine different materials were tested: three lactoses, maltodextrin, two microcrystalline celluloses, sodium chloride, sucrose, and glass beads. Morphology, size, and size distribution, true density, bulk and tap density, angle of repose, flow index, and avalanching behavior were quantified for each excipient by scanning electron microscopy, laser time-of-flight analysis, helium pycnometer, graduated cylinder, fixed-height funnel, Flodex (Hanson Research Corp., Chatsworth, California) method, and AeroFlow (TSI, Inc., St. Paul, Minnesota), respectively. Environmental factors were controlled, and the avalanches were studied at various speeds. RESULTS: The strange-attractor graph obtained at 1 rotation per 120 s showed that it was difficult to appreciate the flowability differences among 3-mm glass beads, lactose 100, and lactose 325. However, plotting the raw data as a relationship of the time between each avalanche and the inverse of speed revealed a characteristic linear slope for each sample. Furthermore, a new flowability index based on the SD calculated from the raw data gave results that were consistent with Carr's index. A cohesive index also can be determined by avalanche behavior, and it reflects the stability of the rapid particular rearrangements of powder. CONCLUSION: A novel method of evaluating avalanche measurements makes it possible to better characterize powder flowability and to predict powder behavior under working conditions.