A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing ...element (intein) from
Saccharomyces cerevisiae (
Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from
Bacillus circulans as an affinity tag. The concept is based on the observation that the modified
Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.
We present measurements of the near-infrared brightness of Io's hot spots derived from 2 to 5 m imaging with adaptive optics on the Keck and Gemini N telescopes. The data were obtained on 271 nights ...between 2013 August and the end of 2018, and include nearly 1000 detections of over 75 unique hot spots. The 100 observations obtained between 2013 and 2015 have been previously published in de Kleer & de Pater the observations since the start of 2016 are presented here for the first time, and the analysis is updated to include the full five-year data set. These data provide insight into the global properties of Io's volcanism. Several new hot spots and bright eruptions have been detected, and the preference for bright eruptions to occur on Io's trailing hemisphere noted in the 2013-2015 data is strengthened by the larger data set and remains unexplained. The program overlapped in time with Sprint-A/EXCEED and Juno observations of the Jovian system, and correlations with transient phenomena seen in other components of the system have the potential to inform our understanding of the impact of Io's volcanism on Jupiter and its neutral/plasma environment.
A survey was designed to obtain information concerning ways clubhouses affiliated with the International Center for Clubhouse Development (ICCD) promote practices that improve the physical health of ...members. This study examined perceptions of the need for health promotion interventions, current and planned health promotion practices, and barriers to change and program development. The mean number of health promotion activities ICCD clubhouses (
N
= 219) report providing was 5.24, SD = 2.42, range = 1 to 10. Despite barriers (e.g., cost), results indicate that every clubhouse responding to this survey offers at least one health promotion activity.
Individuals with serious mental illness (SMI) have significantly greater risk of co-morbid health problems and premature death, and there is need for interventions that can improve physical fitness ...and overall health. Accordingly, a study was conducted which evaluated the effectiveness of a structured physical exercise program that was developed as part of a wellness project in an ICCD Certified Clubhouse. Seventeen clubhouse members completed a 16-week program with evidence of significant improvement in aerobic capacity and perceived mental health as well as positive trends in perceived improvements in physical and social functioning. Qualitative data indicated satisfaction with the program by all participants, especially the value of group support, while also highlighting the need for greater attention to nutrition as part of a future program. Moreover, the study found that a structured exercise program can be successfully provided to members of an ICCD Certified Clubhouse.
Using archival near-infrared observations from the Keck and Lick Observatories and the Hubble Space Telescope, we document the evolution of Neptune’s cloud activity from 1994 to 2022. We calculate ...the fraction of Neptune’s disk that contained clouds, as well as the average brightness of both cloud features and cloud-free background over the planet’s disk. We observe cloud activity and brightness maxima during 2002 and 2015, and minima during 2007 and 2020, the latter of which is particularly deep. Neptune’s lack of cloud activity in 2020 is characterized by a near-total loss of clouds at mid-latitudes and continued activity at the South Pole. We find that the periodic variations in Neptune’s disk-averaged brightness in the near-infrared H (1.6μm), K (2.1μm), FWCH4P15 (893 nm), F953N (955 nm), FWCH4P15 (965 nm), and F845M (845 nm) bands are dominated by discrete cloud activity, rather than changes in the background haze. The clear positive correlation we find between cloud activity and Solar Lyman-Alpha (121.56 nm) irradiance lends support to the theory that the periodicity in Neptune’s cloud activity results from photochemical cloud/haze production triggered by Solar ultraviolet emissions.
•Neptune’s near-infrared cloud activity from 1994 to 2022 exhibits periodic variation.•We show evidence that Neptune’s cloud activity is correlated with Solar activity.•During late 2019 there was a dramatic drop in Neptune’s cloud activity.•From late 2019 onwards, only the south pole exhibited cloud activity.
Using near-infrared observations of Neptune from the Keck and Lick Observatories, and the Hubble Space Telescope in combination with amateur datasets, we calculated the drift rates of prominent ...infrared-bright cloud features on Neptune between 2018 and 2021. These features had lifespans of ∼1 day to ≥1 month and were located at mid-latitudes and near the south pole. Our observations permitted determination of drift rates via feature tracking. These drift rates were compared to three zonal wind profiles describing Neptune’s atmosphere determined from features tracked in H band (1.6 μm), K’ band (2.1 μm), and Voyager 2 data at visible wavelengths. Features near −70deg measured in the F845M filter (845 nm) were particularly consistent with the K’ wind profile. The southern mid-latitudes hosted multiple features whose lifespans were ≥1 month, providing evidence that these latitudes are a region of high stability in Neptune’s atmosphere. We also used HST F467M (467 nm) data to analyze a dark, circumpolar wave at −60° latitude observed on Neptune since the Voyager 2 era. Its drift rate in recent years (2019–2021) is 4.866±0.009°/day. This is consistent with previous measurements by Karkoschka (2011), which predict a 4.858±0.022°/day drift rate during these years. It also gained a complementary bright band just to the north.
•Late 2019 through 2020 showed a decrease in clouds at mid-latitudes.•The South Pole remained active during 2020 as the primary region of cloud activity.•At 1.6 μm a bright ring appeared at −66 degrees latitude.•The South Polar Wave (−60 deg) at 0.467 μm gained a bright band to the north.•The South Polar Wave’s speed is consistent with results from a decade before.
T4 phage polynucleotide kinase (PNK) was identified over 35 years ago and has become a staple reagent for molecular biologists. The enzyme displays 5′-hydroxyl kinase, 3′-phosphatase, and ...2′,3′-cyclic phosphodiesterase activities against a wide range of substrates. These activities modify the ends of nicked tRNA generated by a bacterial response to infection and facilitate repair by T4 RNA ligase. DNA repair enzymes that share conserved motifs with PNK have been identified in eukaryotes. PNK contains two functionally distinct structural domains and forms a homotetramer. The C-terminal phosphatase domain is homologous to the L-2-haloacid dehalogenase family and the N-terminal kinase domain is homologous to adenylate kinase. The active sites have been characterized through structural homology analyses and visualization of bound substrate.
BsoBI is a type II restriction enzyme found in
Bacillus stearothermophilus JN209 that recognizes the symmetric sequence 5′-CYCGRG-3′ (Y=C or T; R=A or G) and cleaves between the first and second base ...to generate a four-base 5′ extension. The cloning and sequencing of
BsoBI restriction-modification system has been described by Ruan et al. Mol. Gen. Genet. 252 (1996) 695–699. Here we report the overexpression of
BsoBI restriction endonuclease gene in
E. coli by insertion of the endonuclease gene into an expression vector pRRS. The recombinant
BsoBI was purified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitutive expression of
BsoBI from pRRS is lethal to
E. coli in the absence of the cognate methylase. The
bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro and transferred into
E. coli via plasmid vectors in the absence of the cognate methylase. Surviving transformants were selected that carry
BsoBI variants which lost endonuclease activity. DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, D246G and E252K can still bind DNA despite the loss of cleavage activity. Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.
T4 phage polynucleotide kinase (PNK) displays 5′‐hydroxyl kinase, 3′‐phosphatase and 2′,3′‐cyclic phosphodiesterase activities. The enzyme phosphorylates the 5′ hydroxyl termini of a wide variety of ...nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single‐stranded (ss)DNA oligonucleotide substrates of various lengths and sequences. In these structures, the 5′ ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases. The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5′‐GT DNA end than for a 5′‐TG end. The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique substrate molecule.
Using near-infrared observations of Neptune from the Keck and Lick Observatories, and the Hubble Space Telescope in combination with amateur datasets, we calculated the drift rates of prominent ...infrared-bright cloud features on Neptune between 2018 and 2021. These features had lifespans of \(\sim 1\) day to \(\geq\)1 month and were located at mid-latitudes and near the south pole. Our observations permitted determination of drift rates via feature tracking. These drift rates were compared to three zonal wind profiles describing Neptune's atmosphere determined from features tracked in H band (1.6 \(\mu m\)), K' band (2.1 \(\mu m\)), and Voyager 2 data at visible wavelengths. Features near \(-70 \deg\) measured in the F845M filter (845nm) were particularly consistent with the K' wind profile. The southern mid-latitudes hosted multiple features whose lifespans were \(\geq\)1 month, providing evidence that these latitudes are a region of high stability in Neptune's atmosphere. We also used HST F467M (467nm) data to analyze a dark, circumpolar wave at \(- 60 \deg\) latitude observed on Neptune since the Voyager 2 era. Its drift rate in recent years (2019-2021) is \(4.866 \pm 0.009 \deg \)/day. This is consistent with previous measurements by Karkoschka (2011), which predict a \(4.858 \pm 0.022 \deg\)/day drift rate during these years. It also gained a complementary bright band just to the north.
PDF
