Cell-specific switch for epileptiform activity: critical role of interneurons in the mouse subicular network.

During epileptic seizures, neuronal network activity is hyper synchronized whereby GABAergic parvalbumin-interneurons may have a key role. Previous studies have mostly utilized 4-aminopyridine to induce epileptiform discharges in brain slices from healthy animals. However, it is not clear if the seizure-triggering ability of parvalbumin-interneurons also holds true without the use of external convulsive agents. Here, we investigate whether synchronized activation of parvalbumin-interneurons or principal cells can elicit epileptiform discharges in subiculum slices of epileptic mice. We found that selective synchronized activation of parvalbumin-interneurons or principal cells with optogenetics do not result in light-induced epileptiform discharges (LIEDs) neither in epileptic nor in normal brain slices. Adding 4-aminopyridine to slices, activation of parvalbumin-interneurons still failed to trigger LIEDs. In contrast, such activation of principal neurons readily generated LIEDs with features resembling afterdischarges. When GABAA receptor blocker was added to the perfusion medium, the LIEDs were abolished. These results demonstrate that in subiculum, selective synchronized activation of principal excitatory neurons can trigger epileptiform discharges by recruiting a large pool of downstream interneurons. This study also suggests region-specific role of principal neurons and interneurons in ictogenesis, opening towards differential targeting of specific brain areas for future treatment strategies tailored for individual patients with epilepsy.

Accuracy Assessment of NLCD 2011 percent impervious cover for selected USA Metropolitan Areas.

The emergence of high-resolution land cover data has created the opportunity to assess the accuracy of impervious cover (IC) provided by the National Land Cover Database (NLCD). We assessed the accuracy of the 900 m2 NLCD2011 %IC for 18 metropolitan areas throughout the conterminous United States using reference data from 1 m2 land cover data developed as part of the United States Environmental Protection Agency's EnviroAtlas project. Agreement was assessed from two perspectives: 1) sensitivity to the size of the assessment unit used for the comparison, and 2) utility of NLCD %IC to serve as a proxy for high-resolution IC. The former perspective was considered because statistical relationships can be sensitive to assessment unit size and shape, and the latter perspective was considered because high resolution (reference) %IC data are not available nationwide. The utility of NLCD %IC as a proxy for the high resolution data was assessed for seven lattice (square) cell sizes ranging from 1 ha to 200 ha using four EnviroAtlas IC indicators: 1) %IC per 100 ha (1 km2); 2) %IC by Census block group; 3) %IC within a 15 m (radius) of the riparian zone, and; 4) %IC within a 50 m (radius) of the riparian zone. Agreement was quantified as per assessment unit deviation (NLCD %IC - reference %IC) and summarized as Mean Absolute Deviation (MAD) and Mean Deviation (MD) both within and across the 18 metropolitan areas. Ordinary least squares (OLS) regression (y = reference %IC and x = NLCD %IC) was also used to evaluate the quality of the NLCD %IC data. MAD was ≤ 5% for six of the seven lattice cell sizes. MAD was also ≤ 5% for Census block groups > 100 ha and for both riparian units. These results suggest that uncertainty attributable to the measurement of %IC was no greater than the uncertainty related to the effect of IC on aquatic resources that have been derived from studies of aquatic condition (e.g., benthic fauna) over a range of %IC. Overall, agreement was variable from one metropolitan area to the next. Agreement improved as assessment unit size increased and declined as the level of urbanization (NLCD %IC) increased. NLCD %IC tended to underestimate reference %IC overall, but NLCD %IC was sometimes greater than reference %IC in urbanized settings.

A guide for evaluating and reporting map data quality: Affirming Shao et al. "Overselling overall map accuracy misinforms about research reliability".

CONTEXT: Landscape ecologists often use thematic map data in their research. Greater familiarity with thematic map accuracy assessment protocols will enhance appropriate use and interpretation of map quality data. OBJECTIVES: Provide an overview of thematic map accuracy assessment protocols and simple, non-quantitative guidelines to assess the quality of the thematic map data that landscape ecologists use in their research. METHODS: Synthesis and interpretation of salient literature on map accuracy assessment. CONCLUSIONS: Landscape ecologists can adopt three simple rules to improve their use and interpretation of map data: 1) use the map quality data only if the accuracy assessment protocols adhere to rigorous, well-established standards for the sampling design, response design, and analysis; 2) focus on class-specific accuracy via user's and producer's accuracies (or the complementary measures commission and omission error rates); and 3) use the criterion that an accuracy assessment that reports class-specific accuracies accompanied by standard errors is a strong indicator of a rigorous assessment.

Influence of high-resolution data on the assessment of forest fragmentation.

CONTEXT: Remote sensing has been a foundation of landscape ecology. The spatial resolution (pixel size) of remotely sensed land cover products has improved since the introduction of landscape ecology in the United States. Because patterns depend on spatial resolution, emerging improvements in the spatial resolution of land cover may lead to new insights about the scaling of landscape patterns. OBJECTIVE: We compared forest fragmentation measures derived from very high resolution (1 m2) data with the same measures derived from the commonly used (30 m × -30 m; 900 m2) Landsat-based data. METHODS: We applied area-density scaling to binary (forest; non-forest) maps for both sources to derive source-specific estimates of dominant (density ≥ 60%), interior (≥ 90%), and intact (100%) forest. RESULTS: Switching from low- to high-resolution data produced statistical and geographic shifts in forest spatial patterns. Forest and non-forest features that were "invisible" at low resolution but identifiable at high resolution resulted in higher estimates of dominant and interior forest but lower estimates of intact forest from the high-resolution source. Overall, the high-resolution data detected more forest that was more contagiously distributed even at larger spatial scales. CONCLUSION: We anticipate that improvements in the spatial resolution of remotely sensed land cover products will advance landscape ecology through reinterpretations of patterns and scaling, by fostering new landscape pattern measurements, and by testing new spatial pattern-ecological process hypotheses.

Spatial patterns of the United States National Land Cover Dataset (NLCD) land-cover change thematic accuracy (2001-2011).

Research on spatial non-stationarity of land-cover classification accuracy has been ongoing for over two decades with most of the work focusing on single date maps. We extend the understanding of thematic map accuracy spatial patterns by: (1) quantifying spatial patterns of map-reference agreement for class-specific land-cover change rather than class-specific land cover for both omission and commission expressions of map error; (2) reporting goodness-of-fit estimates for the empirical models, which have been lacking in previous assessments, and; (3) using the empirical model results to map the locations of the relative likelihoods of map-reference agreement for specific land-cover change classes. We evaluated 10 map-based explanatory variables in single and multivariable logistic regression models to predict the likelihood of agreement between map and reference land-cover change (2001-2011) labels using the National Land Cover Database (NLCD) 2011 land cover and accuracy data. Logistic models for omission error had better goodness-of-fit estimates than models for commission error. For the omission error models, the explanatory variable, density of the mapped class-specific change in the immediate neighbourhood surrounding the sample pixel, produced the best model fit results (Tjur coefficient of discrimination, D, ranged from 0.59 to 0.98) compared to multivariable models and all other single explanatory variable models. Maps of the predicted likelihood of map-reference agreement produced from the best fitting omission error models provide a spatially explicit description of spatial variation of classification uncertainty at both local and regional scales. Application of the models indicated higher likelihoods of agreement (>50%) comprised a greater proportion of the land-cover change class area than the proportion of the land-cover change class with lower likelihoods of agreement. NLCD users can apply reported equations to map land-cover change uncertainty.

Prolonged life of human acute hippocampal slices from temporal lobe epilepsy surgery.

Resected hippocampal tissue from patients with drug-resistant epilepsy presents a unique possibility to test novel treatment strategies directly in target tissue. The post-resection time for testing and analysis however is normally limited. Acute tissue slices allow for electrophysiological recordings typically up to 12 hours. To enable longer time to test novel treatment strategies such as, e.g., gene-therapy, we developed a method for keeping acute human brain slices viable over a longer period. Our protocol keeps neurons viable well up to 48 hours. Using a dual-flow chamber, which allows for microscopic visualisation of individual neurons with a submerged objective for whole-cell patch-clamp recordings, we report stable electrophysiological properties, such as action potential amplitude and threshold during this time. We also demonstrate that epileptiform activity, monitored by individual dentate granule whole-cell recordings, can be consistently induced in these slices, underlying the usefulness of this methodology for testing and/or validating novel treatment strategies for epilepsy.

Accuracy assessment of NLCD 2011 impervious cover data for the Chesapeake Bay region, USA.

The National Land Cover Database (NLCD) contains three eras (2001, 2006, 2011) of percentage urban impervious cover (%IC) at the native pixel size (30 m-×-30 m) of the Landsat Thematic Mapper satellite. These data are potentially valuable to environmental managers and stakeholders because of the utility of %IC as an indicator of watershed and aquatic condition, but lack an accuracy assessment because of the absence of suitable reference data. Recently developed 1 m2 land cover data for the Chesapeake Bay region makes it possible to assess NLCD %IC accuracy for a 262,000 km2 region based on a census rather than a sample of reference data. We report agreement between the two %IC datasets for watersheds and the riparian zones within watersheds and four additional square units. The areas of the six assessment units were 40 ha cell, 433 ha (riparian mean), 2756 ha cell, 5626 ha cell, 8569 ha (watershed mean) and 22,500 ha cell. Mean Absolute Deviation (MAD) and Mean Deviation (MD) were about 1.5% and -1.5%, respectively, for each of the assessment units except for the riparian unit, for which MAD and MD were 0.88 and 0.62, respectively. NLCD reliably reproduced %IC from the 1 m2 data with a small, consistent tendency for underestimation. Results were sensitive to assessment unit choice. The results for the four largest assessment units had very similar regression parameters, R2 values, and bias patterns. Results for the riparian assessment were different from those for the watershed unit and the other three larger units. MAD was about 50% less for the riparian zones than it was for the watersheds, the direction of bias was less consistent, and NLCD %IC was uniformly higher than 1 m2 %IC in urbanized riparian zones. For the smallest unit, bias patterns were more similar to the riparian unit and regression results were more similar to the four larger units. MAD and MD were also sensitive to the amount of urbanization, increasing as NLCD %IC increased. The low overall bias and positive relationship between bias and urbanization suggest that the benefits of obtaining 1 m2 IC data outside of urban areas may not outweigh the costs of obtaining such data.

Exercise-induced dehydration does not alter time trial or neuromuscular performance.

This study examined the effect of exercise-induced dehydration by ~4% body mass loss on 5-km cycling time trial (TT) performance and neuromuscular drive, independent of hyperthermia. 7 active males were dehydrated on 2 occasions, separated by 7 d. Participants remained dehydrated (DEH, -3.8±0.5%) or were rehydrated (REH, 0.2±0.6%) over 2 h before completing the TT at 18-25 °C, 20-30% relative humidity. Neuromuscular function was determined before dehydration and immediately prior the TT. The TT started at the same core temperature (DEH, 37.3±0.3°C; REH, 37.0±0.2 °C (P>0.05). Neither TT performance (DEH, 7.31±1.5 min; REH, 7.10±1.3 min (P>0.05)) or % voluntary activation were affected by dehydration (DEH, 88.7±6.4%; REH, 90.6±6.1% (P>0.05)). Quadriceps peak torque was significantly elevated in both trials prior to the TT (P<0.05), while a 19% increase in the rate of potentiated peak twitch torque development (P<0.05) was observed in the DEH trial only. All other neuromuscular measures were similar between trials. Short duration TT performance and neuromuscular function are not reduced by dehydration, independent of hyperthermia.

The function of neuromuscular compartments in human shoulder muscles.

The aim of this study was to use a surface electromyographic (sEMG) technique with a ballistic isotonic shoulder joint adduction movement to determine the function of the neuromuscular compartments (NMCs) within the pectoralis major, deltoid, and latissimus dorsi muscles. Sixteen male subjects (mean age 22 yr) with no known history of shoulder pathologies volunteered to participate. Timing and intensity of muscle contraction, recorded with 15 pairs of bipolar sEMG electrodes, were compared during performance of 40° coronal-plane ballistic [movement time (MT) < 400 ms] shoulder joint adduction movements. The results suggested that heterogeneous sEMG was present across the breadth of all three muscles, indicating the presence of individual NMCs with significant (P < 0.05) differences observed within the three muscles in NMC onset, duration, timing of peak NMC intensity, or relative intensity of NMC activation. For example, within the deltoid NMC activation was closely related to moment arm (MA) length with the NMC, with the largest antagonist MA deltoid NMC3 having a late period of activation [antagonist (Ant)] to slow glenohumeral joint (GHJ) rotation and maintain its final joint position [with agonist 2 burst (Ag2)]. The most obvious triphasic EMG patterns (e.g., Ag1-Ant-Ag2) were observed between the first NMCs activated in the two agonist muscles and the last NMC activated in the antagonist deltoid muscle. In conclusion, our findings suggest the presence of in-parallel NMCs within the superficial muscles of the GHJ and show that biomechanical parameters, such as the MA at end-point movement position, influence the function of each NMC and its contribution to alternating patterns of agonist and antagonist muscle activity typical of ballistic movement.

Muscles within muscles: Coordination of 19 muscle segments within three shoulder muscles during isometric motor tasks.

The aim of the present study was to determine how the intra-muscular segments of three shoulder muscles were coordinated to produce isometric force impulses around the shoulder joint and how muscle segment coordination was influenced by changes in movement direction, mechanical line of action and moment arm (ma). Twenty male subjects (mean age 22 years; range 18-30 years) with no known history of shoulder pathologies, volunteered to participate in this experiment. Utilising an electromyographic technique, the timing and intensity of contraction within 19 muscle segments of three superficial shoulder muscles (Pectoralis Major, Deltoid and Latissimus Dorsi) were studied and compared during the production of rapid (e.g. approximately 400ms time to peak) isometric force impulses in four different movement directions of the shoulder joint (flexion, extension, abduction and adduction). The results of this investigation have suggested that the timing and intensity of each muscle segment's activation was coordinated across muscles and influenced by the muscle segment's moment arm and its mechanical line of action in relation to the intended direction of shoulder movement (e.g. flexion, extension, abduction or adduction). There was also evidence that motor unit task groups were formed for individual motor tasks which comprise motor units from both adjacent and distant muscles. It was also confirmed that for any particular motor task, individual muscle segments can be functionally classified as prime mover, synergist or antagonist - classifications which are flexible from one movement to the next.

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells.

MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC(50) that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.

Anatomical and possible clinical relationships between the calcaneofibular ligament and peroneus brevis--a pilot study.

The authors have observed in their clinical practice patients presenting with chronic retromalleolar pain following lateral ankle injuries. It has been hypothesized that persistent retromalleolar pain following a supination sprain may be due to peroneus brevis (PB) tendon tears (Boruta et al. 1990). The aims of this study were to investigate whether an anatomical relationship exists between the calcaneofibular ligament (CFL) and PB, and if so, the significance of this relationship in the positions of supination sprain and talar tilt test. Seven out of eight cadaveric ankles demonstrated fibrous connecting tissue between the tendon of PB and CFL. Four of the eight ankles demonstrated PB tendon abnormalities. The presence of connecting tissue between CFL and PB suggests an anatomical basis for concomitant damage to the PB tendon with a supination sprain, thus supporting the hypothesis that there may be an anatomical basis for persistent retromalleolar pain subsequent to injury to the lateral ankle complex.

Uptake and incorporation of docosahexaenoic acid (DHA) into neuronal cell body and neurite/nerve growth cone lipids: evidence of compartmental DHA metabolism in nerve growth factor-differentiated PC12 cells.

Docosahexaenoic acid (DHA) accumulates in nerve endings of the brain during development. It is released from the membrane during ischemia and electroconvulsive shock. DHA optimizes neurologic development, it is neuroprotective, and rat adrenopheochromocytoma (PC12) cells have decreased PLA2 activity when DHA is present. To characterize DHA metabolism in PC12 cells, media were supplemented with [3H]DHA or [3H]glycerol. Fractions of nerve growth cone particles (NGC) and cell bodies were prepared and the metabolism of the radiolabeled substrates was determined by thin-layer chromatography. [3H]glycerol incorporation into phospholipids indicated de novo lipid synthesis. [3H]DHA uptake was more rapid in the cell bodies than in the NGC. [3H]DHA first esterified in neutral lipids and later in phospholipids (phosphatidylethanolamine). [3H]glycerol primarily labeled phosphatidylcholine. DHA uptake was compartmentalized between the cell body and the NGC. With metabolism similar to that seen in vivo, PC12 cells are an appropriate model to study DHA in neurons.

Shape control of CdSe nanocrystals

Nanometre-size inorganic dots, tubes and wires exhibit a wide range of electrical and optical properties that depend sensitively on both size and shape, and are of both fundamental and technological interest. In contrast to the syntheses of zero-dimensional systems, existing preparations of one-dimensional systems often yield networks of tubes or rods which are difficult to separate. And, in the case of optically active II-VI and III-V semiconductors, the resulting rod diameters are too large to exhibit quantum confinement effects. Thus, except for some metal nanocrystals, there are no methods of preparation that yield soluble and monodisperse particles that are quantum-confined in two of their dimensions. For semiconductors, a benchmark preparation is the growth of nearly spherical II-VI and III-V nanocrystals by injection of precursor molecules into a hot surfactant. Here we demonstrate that control of the growth kinetics of the II-VI semiconductor cadmium selenide can be used to vary the shapes of the resulting particles from a nearly spherical morphology to a rod-like one, with aspect ratios as large as ten to one. This method should be useful, not only for testing theories of quantum confinement, but also for obtaining particles with spectroscopic properties that could prove advantageous in biological labelling experiments and as chromophores in light-emitting diodes.

A balance of signaling by Rho family small GTPases RhoA, Rac1 and Cdc42 coordinates cytoskeletal morphology but not cell survival.

Rho family GTPases are known to be involved in cytoskeletal reorganization. We examined the possibility that these functions may be dictated by a balance of Rho family GTPase signaling. Using transient viral expression of RhoA, Rac1, Cdc42 and their mutants, as well as C3 exoenzyme, we altered cytoskeletal organization under normal growth conditions. Overexpression of wild-type or constitutively active forms of the Rho family GTPases led to their respective activation phenotypes. Overexpression of dominant negative forms of given Rho family GTPases led to a phenotype consistent with activation of the other Rho family GTPase. Treatment with C. difficile toxin A, that inactivates all Rho family GTPases, led to the transient appearance of a variety of activation phenotypes. Previously, we reported that inactivation of Rho led to induction of apoptosis, implying that Rho may play an important role in cell survival signaling. This signaling, however, is not affected by expression of any forms of Rac1 or Cdc42, and only inactivation of Rho led to induction of apoptosis. Rho family GTPases appear to coordinate cytoskeletal organization by a balance of signaling, while cell survival is regulated by a distinct Rho-mediated signaling pathway.

Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells.

Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation.

Muscles within muscles: the neuromotor control of intra-muscular segments.

The aim of this investigation was to anatomically identify, and then determine the function of, individual segments within the human deltoid muscle. The anatomical structure of the deltoid was determined through dissection and/or observation of the shoulder girdles of 11 male cadavers (aged 65-84 years). These results indicate that the deltoid consists of seven anatomical segments (D1-D7) based upon the distinctive arrangement of each segment's origin and insertion. Radiographic analysis of a cadaveric shoulder joint suggested that only the postero-medial segment D7 has a line of action directed below the shoulder joint's axis of rotation. The functional role of each individual segment was then determined utilising an electromyographic (EMG) technique. Seven miniature (1 mm active plate; 7 mm interelectrode distance) bipolar surface electrodes were positioned over the proximal portion of each segment's muscle belly in 18 male and female subjects (18-30 years). EMG waveforms were then recorded during the production of rapid isometric shoulder abduction and adduction force impulses with the shoulder joint in 40 degrees of abduction in the plane of the scapula. Each subject randomly performed 15 abduction and 15 adduction isometric force impulses following a short familiarisation period. All subjects received visual feed back on the duration and amplitude of each isometric force impulse produced via a visual force-time display which compared subject performance to a criterion force-time curve. Movement time was 400 ms (time-to-peak isometric force) at an intensity level of 50% maximal voluntary contraction. Temporal and intensity analyses of the EMG waveforms, as well as temporal analysis of the isometric force impulses, revealed the neuromotor control strategies utilised by the CNS to control the activity of each muscle segment. The results showed that segmental neuromotor control strategies differ across the breadth of the muscle and that individual segments of the deltoid can be identified as having either "prime mover", "synergist", "stabiliser" or "antagonist" functions; functional classifications normally associated with whole muscle function. Therefore, it was concluded that the CNS can "fine tune" the activity of at least six discrete segments within the human deltoid muscle to efficiently meet the demands of the imposed motor task.

Differential requirements for basic amino acids in transcription factor IIIA-5S gene interaction.

Basic amino acids Arg, Lys, and His in the Cys2His2 zinc fingers of transcription factor IIIA (TFIIIA) potentially have important roles in factor binding to the extended internal control region (ICR) of the 5S ribosomal gene. Conserved and non-conserved basic residues in the N-terminal fingers I, II, III and the more C-terminal fingers V and IX were analyzed by site-directed mutagenesis and DNase I protection in order to assess their individual requirement in the DNA-binding mechanism. In the DNA recognition helix of finger II, the conserved Arg at position 62 (N-terminal side of the first zinc-coordinating histidine) was changed to a Leu or Gln. Both the R62L and R62Q mutations inhibited Xenopus TFIIIA-dependent DNase I footprinting along the entire 5S gene ICR. When His-58 (non-conserved basic residue with DNA-binding potential in the same helical region) was changed to a Gln, the mutated protein was able to protect the ICR from DNase I digestion. Therefore, Arg-62 is individually required for TFIIIA binding over the entire ICR whereas His-58 is not. Fingers V and IX have conserved Arg residues in positions identical to Arg-62 in finger II (Arg-154 in finger V and Arg-271 in finger IX). When these residues were changed to Leu and Ile respectively, TFIIIA-dependent DNase I protection was observed along the entire 5S gene ICR. These results indicate differing DNA-binding mechanisms by the N-terminal fingers versus the C-terminal fingers at the level of individual amino acid-nucleotide interactions. In the N-terminal finger I, the conserved Lys at position 11 outside the recognition helix and a conserved hydrophobic Trp at position 28 within the helix were changed to an Ala and Ser respectively. The K11A change inhibited TFIIIA-dependent DNase I protection to a much greater extent than the W28S change.