Does experience in talking facilitate speech repetition?

Speech is unique among highly skilled human behaviors in its ease of acquisition by virtually all individuals who have normal hearing and cognitive ability. Vocal imitation is essential for acquiring speech, and it is an important element of social communication. The extent to which age-related changes in cognitive and motor function affect the ability to imitate speech is poorly understood. We analyzed the distributions of response times (RT) for repeating real words and pseudowords during fMRI. The average RT for older and younger participants was not different. In contrast, detailed analysis of RT distributions revealed age-dependent differences that were associated with changes in the time course of the BOLD response and specific patterns of regional activation. RT-dependent activity was observed in the bilateral posterior cingulate, supplementary motor area, and corpus callosum. This approach provides unique insight into the mechanisms associated with changes in speech production with aging.

Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line.

Intermediate-conductance K(+) (Kcnn4) channels in the apical and basolateral membranes of epithelial cells play important roles in agonist-induced fluid secretion in intestine and colon. Basolateral Kcnn4 channels have been well characterized in situ using patch-clamp methods, but the investigation of Kcnn4 channels in apical membranes in situ has been hampered by a layer of mucus that prevents seal formation. In the present study, we used patch-clamp methods to characterize Kcnn4 channels in the apical membrane of IEC-18 cells, a cell line derived from rat small intestine. A monolayer of IEC-18 cells grown on a permeable support is devoid of mucus, and tight junctions enable selective access to the apical membrane. In inside-out patches, Ca(2+)-dependent K(+) channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca(2+)-activated K(+) channel blocker) and apamin (a Kcnn1-3/small-conductance, Ca(2+)-activated K(+) channel blocker) present in the pipette solution exhibited a single-channel conductance of 31 pS with inward rectification. The currents were reversibly blocked by TRAM-34 (a Kcnn4 blocker) with an IC(50) of 8.7 ± 2.0 µM. The channels were not observed when charybdotoxin, a peptide inhibitor of Kcnn4 channels, was added to the pipette solution. TRAM-34 was less potent in inhibiting Kcnn4 channels in patches from apical membranes than in patches from basolateral membranes, which was consistent with a preferential expression of Kcnn4c and Kcnn4b isoforms in apical and basolateral membranes, respectively. The expression of both isoforms in IEC-18 cells was confirmed by RT-PCR and Western blot analyses. This is the first characterization of Kcnn4 channels in the apical membrane of intestinal epithelial cells.

A KLF4-miRNA-206 autoregulatory feedback loop can promote or inhibit protein translation depending upon cell context.

Krüppel-like factor 4 (KLF4), a transcription factor that regulates cell fate in a context-dependent fashion, is normally induced upon growth arrest or differentiation. In many cancer cells there is dysregulation, with increased expression in proliferating cells. To identify sequence elements that mediate KLF4 suppression in normal epithelial cells, we utilized a luciferase reporter and RK3E cells, which undergo a proliferation-differentiation switch to form an epithelial sheet. A translational control element (TCE) within the KLF4 3'-untranslated region interacted with microRNAs (miRs) 206 and 344-1 to promote or inhibit KLF4 expression, respectively, in proliferating epithelial cells. Overall, the TCE suppressed expression in proliferating primary human mammary epithelial cells, but this suppressive effect was attenuated in immortalized mammary epithelial MCF10A cells, in which Dicer1 and miR-206 promoted KLF4 expression and TCE reporter activity. In contrast to MCF10A cells, in breast cancer cells the activity of miR-206 was switched, and it repressed KLF4 expression and TCE reporter activity. As miR-206 levels were KLF4 dependent, the results identify a KLF4-miR-206 feedback pathway that oppositely affects protein translation in normal cells and cancer cells. In addition, the results indicate that two distinct miRs can have opposite and competing effects on translation in proliferating cells.

Constitutive, translation-independent opening of the protein-conducting channel in the endoplasmic reticulum.

Secretory and membrane proteins are translocated into the endoplasmic reticulum (ER) through a translocon assembled as a tetramer of Sec61 protein-conducting channels (PCC). How the opening of the PCCs in the tetramer is regulated through the protein translocation cycle is poorly understood. In this study, the permeability of PCCs in native ER membranes to small molecules was measured using fluorescence and electrophysiological techniques. Although the PCCs were closed at 4 degrees C, they were constitutively open at physiological temperatures in the absence of protein translation or a bound ribosome. The open PCCs occurred in clusters that are likely to correspond to the simultaneous opening of three or four PCCs in a translocon. The binding of 60S subunits to a ribosome-free membrane increased the number of open PCCs but did not increase the single-channel conductance. The translation-independent, constitutive opening of Sec61 PCCs provides new insight into the role of the translocon in the transport of small molecules across the ER membrane.

Resistance training increases heat shock protein levels in skeletal muscle of young and old rats.

Heat shock proteins (HSP) HSP72, HSC70 and HSP25 protein levels and mRNA levels of HSP72 genes (Hsp72-1, Hsp72-2, Hsp72-3) and HSC70 were examined in tibialis anterior muscles from young and old rats following 4.5 weeks of heavy resistance exercise. Young (3 months) (n=10) and old (30 months) (n=9) rats were subjected to 14 sessions of electrically evoked resistance training using stretch-shortening contractions of the left limb that activated the dorsiflexor muscle group, including the tibialis anterior muscle, while the right side served as the intra-animal control. Muscle wet weight of the left tibialis anterior increased by 15.6% in young animals compared to the untrained right side, while the aged rats demonstrated no significant hypertrophy based on muscle wet weight. There were no differences in mRNA expression between the control and experimental muscles in either the old or the young animals for any of the four genes examined. On the other hand, HSP72 levels as determined by Western blots were significantly (p<0.01) higher (968.8 and 409.1%) in the trained as compared to the contralateral control muscle in young and old animals, respectively. HSP25 expression was increased significantly (p<0.01) by training in muscles of young rats (943.1%) and old rats (420.3%). Moreover, there was no training by age interaction for HSP72, while a significant age and training by age effects were found in muscles for HSP25. There was no change in HSC70 protein expression in response to the training intervention in either age group. SOD-1 enzyme level increased by 66.6% in the trained muscles of the young rats, while this enzyme was 33% lower in trained muscles compared to the untrained control side in old rats. Moreover, a significant (p<0.05) training by age interaction was found for SOD-1 enzyme levels. This study suggests that fast contracting muscles in young and old animals are capable of increasing HSP expression in response to high intensity contractile stress. Furthermore, the data are consistent with the hypothesis that higher levels of oxidative stress in muscles of old animals limit HSP levels and/or function in response to high intensity contractile stress.

The permeability of the endoplasmic reticulum is dynamically coupled to protein synthesis.

Proteins synthesized by the rough endoplasmic reticulum (RER) co-translationally cross the membrane through the pore of a ribosome-bound translocon (RBT) complex. Although this pore is also permeable to small molecules, it is generally thought that barriers to their permeation prevent the cyclical process of protein translation from affecting the permeability of the RER. We tested this hypothesis by culturing Chinese hamster ovary-S cells with inhibitors of protein translation that affect the occupancy of RBTs by nascent proteins and then permeabilizing the plasma membrane and measuring the permeability of the RER to a small molecule, 4-methyl-umbelliferyl-alpha-d-glucopyranoside (4-MalphaG). The premature or normal release of nascent proteins by puromycin or pactamycin, respectively, increased the permeability of the RER to 4-MalphaG by 20-30%. In contrast, inhibition of elongation and the release of nascent proteins by cycloheximide did not increase the permeability, but it prevented the increase in permeability by pactamycin. We conclude that the permeability of the RER is coupled to protein translation by a simple gating mechanism whereby a nascent protein blocks the pore of a RBT during translation, but after release of the nascent protein the pore is permeable to small molecules as long as an empty ribosome remains bound to the translocon.