Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.

Coexistence of linear zones and pinwheels within orientation maps in cat visual cortex

Revealing the layout of cortical maps is important both for understanding the processes involved in their development and for uncovering the mechanisms underlying neural computation. The typical organization of orientation maps in the cat visual cortex is radial; complete orientation cycles are mapped around orientation singularities. In contrast, long linear zones of orientation representation have been detected in the primary visual cortex of the tree shrew. In this study, we searched for the existence of long linear sequences and wide linear zones within orientation preference maps of the cat visual cortex. Optical imaging based on intrinsic signals was used. Long linear sequences and wide linear zones of preferred orientation were occasionally detected along the border between areas 17 and 18, as well as within area 18. Adjacent zones of distinct radial and linear organizations were observed across area 18 of a single hemisphere. However, radial and linear organizations were not necessarily segregated; long (7.5 mm) linear sequences of preferred orientation were found embedded within a typical pinwheel-like organization of orientation. We conclude that, although the radial organization is dominant, perfectly linear organization may develop and perform the processing related to orientation in the cat visual cortex.

Dynamics and orientation of N+(CD3)3-bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor

Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N+(CD3)3-BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42° in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.

Interactions between Senescence and Leaf Orientation Determine in Situ Patterns of Photosynthesis and Photoinhibition in Field-Grown Rice1

Photosynthesis and photoinhibition in field-grown rice (Oryza sativa L.) were examined in relation to leaf age and orientation. Two varieties (IR72 and IR65598-112-2 [BSI206]) were grown in the field in the Philippines during the dry season under highly irrigated, well-fertilized conditions. Flag leaves were examined 60 and 100 d after transplanting. Because of the upright nature of 60-d-old rice leaves, patterns of photosynthesis were determined by solar movements: light falling on the exposed surface in the morning, a low incident angle of irradiance at midday, and light striking the opposite side of the leaf blade in the afternoon. There was an early morning burst of CO2 assimilation and high levels of saturation of photosystem II electron transfer as incident irradiance reached a maximum level. However, by midday the photochemical efficiency increased again almost to maximum. Leaves that were 100 d old possessed a more horizontal orientation and were found to suffer greater levels of photoinhibition than younger leaves, and this was accompanied by increases in the de-epoxidation state of the xanthophyll cycle. Older leaves had significantly lower chlorophyll content but only slightly diminished photosynthesis capacity.

Domains of a Transit Sequence Required for in Vivo Import in Arabidopsis Chloroplasts1

Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.

Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1

We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF1-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-γ accumulation in vivo. The CF1-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-γ.

Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain.

We have isolated a new Drosophila mutant, satori (sat), the males of which do not court or copulate with female flies. The sat mutation comaps with fruitless (fru) at 91B and does not rescue the bisexual phenotype of fru, indicating that sat is allelic to fru (fru(sat)). The fru(sat) adult males lack a male-specific muscle, the muscle of Lawrence, as do adult males with other fru alleles. Molecular cloning and analyses of the genomic and complementary DNAs indicated that transcription of the fru locus yields several different transcripts. The sequence of fru cDNA clones revealed a long open reading frame that potentially encodes a putative transcription regulator with a BTB domain and two zinc finger motifs. In the 5' noncoding region, three putative transformer binding sites were identified in the female transcript but not in male transcripts. The fru gene is expressed in a population of brain cells, including those in the antennal lobe, that have been suggested to be involved in determination of male sexual orientation. We suggest that fru functions downstream of tra in the sex-determination cascade in some neural cells and that inappropriate sexual development of these cells in the fru mutants results in altered sexual orientation of the fly.

Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit.

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase necessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mutant Rna1-1p and, under appropriate physiological conditions, a small portion of the wild-type Rna1p cofractionate with yeast nuclei. Subnuclear fractionation studies show that most of the Rna1p is tightly associated with nuclear components, and that a portion of the active protein can be solubilized by treatments that fail to solubilize inactive Rna1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells. By indirect immuno-fluorescence we show that wild-type Rna1p has three subcellular locations. The majority of the protein is distributed throughout the cytosol, but a portion of the protein is nucleus-associated, located at both the cytosolic surface and within the nucleoplasm. Mutant Rna1-1p is found at the outer nuclear surface and in the cytosol. We propose that a small pool of the wild-type Rna1p is located in the nuclear interior, supporting the model that the same components of the Ran/TC4 GTPase cycle exist on both sides of the nuclear membrane.

Centromere mapping and orientation of the molecular linkage map of rice (Oryza sativa L.).

Rice has become a model cereal plant for molecular genetic research. Rice has the most comprehensive molecular linkage maps with more than 2000 DNA markers and shows synteny and colinearity with the maps of other cereal crops. Until now, however, no information was available about the positions of centromeres and arm locations of markers on the molecular linkage map. Secondary and telotrisomics were used to assign restriction fragment length polymorphism markers to specific chromosome arms and thereby to map the positions of centromeres. More than 170 restriction fragment length polymorphism markers were assigned to specific chromosome arms through gene dosage analysis using the secondary and telotrisomics and the centromere positions were mapped on all 12 linkage groups. The orientations of seven linkage groups were reversed to fit the "short arm on top" convention and the corrected map is presented.

RNA polymerase II/III transcription specificity determined by TATA box orientation.

The TATA box sequence in eukaryotes is located about 25 bp upstream of many genes transcribed by RNA polymerase II (Pol II) and some genes transcribed by RNA polymerase III (Pol III). The TATA box is recognized in a sequence-specific manner by the TATA box-binding protein (TBP), an essential factor involved in the initiation of transcription by all three eukaryotic RNA polymerases. We have investigated the recognition of the TATA box by the Pol II and Pol III basal transcription machinery and its role in establishing the RNA polymerase specificity of the promoter. Artificial templates were constructed that contained a canonical TATA box as the sole promoter element but differed in the orientation of the 8-bp TATA box sequence. As expected, Pol II initiated transcription in unfractionated nuclear extracts downstream of the "forward" TATA box. In distinct contrast, transcription that initiated downstream of the "reverse" TATA box was carried out specifically by Pol III. Importantly, this effect was observed regardless of the source of the DNA either upstream or downstream of the TATA sequence. These findings suggest that TBP may bind in opposite orientations on Pol II and Pol III promoters and that opposite, yet homologous, surfaces of TBP may be utilized by the Pol II and Pol III basal machinery for the initiation of transcription.

Theory of orientation tuning in visual cortex.

The role of intrinsic cortical connections in processing sensory input and in generating behavioral output is poorly understood. We have examined this issue in the context of the tuning of neuronal responses in cortex to the orientation of a visual stimulus. We analytically study a simple network model that incorporates both orientation-selective input from the lateral geniculate nucleus and orientation-specific cortical interactions. Depending on the model parameters, the network exhibits orientation selectivity that originates from within the cortex, by a symmetry-breaking mechanism. In this case, the width of the orientation tuning can be sharp even if the lateral geniculate nucleus inputs are only weakly anisotropic. By using our model, several experimental consequences of this cortical mechanism of orientation tuning are derived. The tuning width is relatively independent of the contrast and angular anisotropy of the visual stimulus. The transient population response to changing of the stimulus orientation exhibits a slow "virtual rotation." Neuronal cross-correlations exhibit long time tails, the sign of which depends on the preferred orientations of the cells and the stimulus orientation.

Mobility and Aging in Denver, Colorado: Travel Behavior, Mobility Barriers, and Perceptions of Transit

The number of seniors in the U.S. today is growing rapidly because of longer life expectancies and the aging Baby Boomer generation. This age groups' travel behavior will have substantial impacts on transportation, economics, safety, and the environment. This research used a mixed-methods approach to address issues of mobility and aging in Denver, Colorado. A quantitative approach was used to answer broad questions about travel behavior and the effects of age, gender, work status, disability, residential location and socio-economic status on mobility. Qualitative interviews with seniors in the Denver metro area were conducted to identify barriers to mobility, decision-making processes and travel decisions, and seniors' perceptions of public transit. The results of the quantitative and qualitative analyses show that residential location is an important variable for determining seniors' travel behaviors and transportation options. Perceptions of public transit were positive, but accessibility and information barriers exist that prevent older adult from using transit. The findings of this study will help to provide transportation and service recommendations to policymakers and planners in the Denver area as well as to inform studies of other North American cities with large aging populations.

The Impact of Transit Oriented Development on the Travel Behaviors of Workers in Denver, Colorado

To combat unsustainable transportation systems characterized by reliance on petroleum, polluting emissions, traffic congestion and suburban sprawl, planners encourage mixed use, densely populated areas that provide individuals with opportunities to live, work, eat and shop without necessarily having to drive private automobiles to accommodate their needs. Despite these attempts, the frequency and duration of automobile trips has consistently increased in the United States throughout past decades. While many studies have focused on how residential proximity to transit influences travel behavior, the effect of workplace location has largely been ignored. This paper asks, does working near a TOD influence the travel behaviors of workers differently than workers living near a TOD? We examine the non-work travel behaviors of workers based upon their commuting mode and proximity to TODs. The data came from a 2009 travel behavior survey by the Denver Regional Council of Governments, which contains 8,000 households, 16,000 individuals, and nearly 80,000 trips. We measure sustainable travel behaviors as reduced mileage, reduced number of trips, and increased use of non-automobile transportation. The results of this study indicate that closer proximity of both households and workplaces to TODs decrease levels of car commuting and that non-car commuting leads to more sustainable personal travel behaviors characterized by more trips made with alternative modes.

Opportunities and Challenges for the Widespread Implementation of Battery Powered Electric Transit Buses

The need for more sustainable public transportation choices drives innovation and provides opportunity for improvement in options. Transit buses provide many advantages for efficient transportation and electric drive vehicles are anticipated to play an increasing role in future transportation systems. A lifecycle cost analysis of battery electric transit buses indicates rate structures and demand charges do not currently have a large impact on lifecycle cost for small fleets of battery electric buses. As fleets grow, policies and rate structures will need to adjust to avoid becoming a barrier to adoption. Battery electric transit buses are now being developed which promise to address the primary issues of high life cycle cost, low reliability, range, and flexibility.