Gα<Subscript>16</Subscript> interacts with tetratricopeptide repeat 1 (TPR1) through its β3 region to activate Ras independently of phospholipase Cβ signaling

Background  

G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα₁₆ as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase Cβ (PLCβ).     

Lactoferricin mediates anti‐inflammatory and anti‐catabolic effects via inhibition of IL‐1 and LPS activity in the intervertebral disc

The catabolic cytokine interleukin‐1 (IL‐1) and endotoxin lipopolysaccharide (LPS) are well‐known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL‐1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti‐catabolic and anti‐inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL‐1 and LPS‐mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL‐1 and LPS‐mediated proteoglycan (PG) depletion, matrix‐degrading enzyme production, and enzyme activity in long‐term (alginate beads) and short‐term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL‐1 and LPS‐mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage‐degrading enzymes, including MMP‐1, MMP‐3, MMP‐13, ADAMTS‐4, and ADAMTS‐5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor‐induced stimulation of oxidative and inflammatory factors such as iNOS, IL‐6, and toll‐like receptor‐2 (TLR‐2) and TLR‐4. Finally, the ability of LfcinB to antagonize IL‐1 and LPS‐mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. J. Cell. Physiol. 228: 1884–1896, 2013. © 2013 Wiley Periodicals, Inc.     

Novel protein fold discovered in the PabI family of restriction enzymes

Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction–modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel β-sheet that forms a ‘half pipe’. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg²⁺. These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.     

Monitoring demographics of a commercially exploited population of shovelnose sturgeon in the Wabash River,Illinois/Indiana,USA

Shovelnose sturgeon (Scaphirhynchus platorynchus, Rafinesque, 1820) in the Wabash River, Illinois/Indiana, USA, provide an important recreational sport and commercial caviar fishery. In fact, it is one of the last commercially viable populations for sturgeon roe harvest. Due to increased demand in the caviar trade and endangered species legislation that protect shovelnose sturgeon in only a portion of their range, efforts of the roe harvest market may continue to divert toward unprotected populations like the shovelnose sturgeon in the Wabash River. Previous studies have shown that increased harvest pressure in this species can affect the age‐at‐maturation and result in recruitment overfishing. Therefore, it is important to closely and continuously monitor commercially exploited populations. Over the past decade (2007–2016), 13,170 shovelnose sturgeon were sampled with boat electroshocking, hoop nets, drift nets, trotlines, and benthic electrified trawls. Captured fish ranged from 61 to 910 mm fork length (FL; mean = 668 mm), with very few fish less than 550 mm FL. Although fish were found to be in a healthy condition (mean relative weight = 87), there was a decrease in the mean condition over time. In addition, we saw declines in mean FL, weight of roe‐per‐fish, and size‐at‐maturity for female fish directly impacted by harvest. The decline of these population parameters, coupled with an increase in total annual mortality and a truncated age frequency distribution, suggest that harvest is negatively impacting the demographics and recruitment of shovelnose sturgeon in the Wabash River. Considering the downward trajectory of population dynamics and high estimates of mortality, their resiliency to continued harvest and environmental changes will be limited.     

Effective control of Schistosoma haematobium infection in a Ghanaian community following installation of a water recreation area

Background

Urogenital schistosomiasis caused by Schistosoma haematobium was endemic in Adasawase, Ghana in 2007. Transmission was reported to be primarily through recreational water contact.

Methods

We designed a water recreation area (WRA) to prevent transmission to school-aged children. The WRA features a concrete pool supplied by a borehole well and a gravity-driven rainwater collection system; it is 30 m² and is split into shallow and deep sections to accommodate a variety of age groups. The WRA opened in 2009 and children were encouraged to use it for recreation as opposed to the local river. We screened children annually for S. haematobium eggs in their urine in 2008, 2009, and 2010 and established differences in infection rates before (2008–09) and after (2009–10) installation of the WRA. After each annual screening, children were treated with praziquantel and rescreened to confirm parasite clearance.

Principal Findings

Initial baseline testing in 2008 established that 105 of 247 (42.5%) children were egg-positive. In 2009, with drug treatment alone, the pre-WRA annual cumulative incidence of infection was 29 of 216 (13.4%). In 2010, this incidence rate fell significantly (p<0.001, chi-squared) to 9 of 245 (3.7%) children after installation of the WRA. Logistic regression analysis was used to determine correlates of infection among the variables age, sex, distance between home and river, minutes observed at the river, low height-for-age, low weight-for-age, low Body Mass Index (BMI)-for-age, and previous infection status.

Conclusion/Significance

The installation and use of a WRA is a feasible and highly effective means to reduce the incidence of schistosomiasis in school-aged children in a rural Ghanaian community. In conjunction with drug treatment and education, such an intervention can represent a significant step towards the control of schistosomiasis. The WRA should be tested in other water-rich endemic areas to determine whether infection prevalence can be substantially reduced.     

Frankenswine,or bringing home the bacon: How close are we to clinical trials in xenotransplantation?

Xenotransplantation—specifically from pig into human—could resolve the critical shortage of organs, tissues and cells for clinical transplantation. Genetic engineering techniques in pigs are relatively well-developed and to date have largely been aimed at producing pigs that either (1) express high levels of one or more human complement-regulatory protein(s), such as decay-accelerating factor or membrane cofactor protein, or (2) have deletion of the gene responsible for the expression of the oligosaccharide, Galα1,3Gal (Gal), the major target for human anti-pig antibodies, or (3) have both manipulations. Currently the transplantation of pig organs in adequately-immunosuppressed baboons results in graft function for periods of 2–6 months (auxiliary hearts) and 2–3 months (life-supporting kidneys). Pig islets have maintained normoglycemia in diabetic monkeys for >6 months. The remaining immunologic barriers to successful xenotransplantation are discussed, and brief reviews made of (1) the potential risk of the transmission of an infectious microorganism from pig to patient and possibly to the public at large, (2) the potential physiologic incompatibilities between a pig organ and its human counterpart, (3) the major ethical considerations of clinical xenotransplantation, and (4) the possible alternatives that compete with xenotransplantation in the field of organ or cell replacement, such as mechanical devices, tissue engineering, stem cell biology and organogenesis. Finally, the proximity of clinical trials is discussed. Islet xenotransplantation is already at the stage where clinical trials are actively being considered, but the transplantation of pig organs will probably require further genetic modifications to be made to the organ-source pigs to protect their tissues from the coagulation/anticoagulation dysfunction that plays a significant role in pig graft failure after transplantation in primates.Key words: islets, pancreatic, genetic engineering, organogenesis, pig, xenotransplantation     

Development of a prodrug of hydantoin based TACE inhibitor

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors led to fused bi-heteroaryl hydantoin series that demonstrate sub-nanomolar potency (Ki) as well as excellent activity in human whole blood (hWBA). However, lead compound 2 posed some formulation challenges which prevented it for further development. A prodrug approach was investigated to address this issue. The pivalate prodrug 3 can be formulated as stable neutral form and demonstrated improved DMPK properties when compared with parent compound.     

Thermochemistry of aqueous solutions of alkylated nucleic acid bases. IV. Enthalpies of hydration of 5-alkyluracils

Enthalpies of sublimation, DeltaH degrees (subl) and of solution in water, DeltaH degrees (sol) were determined for a series of crystalline 1,3-dimethyl-uracil derivatives substituted at the C5-ring carbon atom with alkyl groups (-C(n)H(2n+1), n = 2-4) and some of their C(5.6)-cyclooligomethylene analogues (-(CH2)(n)-, n = 3-5). From these data. enthalpies of hydration DeltaH degrees (hydr)= DeltaH degrees (sol) - DeltaH degrees (subl) were calculated and corrected for energies of cavity formation in pure liquid water in order to obtain enthalpies of interaction, DeltaH degrees (int) of the solutes with their hydration shells. The latter are discussed together with the recalculated DeltaH degrees (int) for variously methylated uracils, obtained previously according to a simplified correction procedure, in terms of perturbations in the energy and scheme of hydration of the diketopyrimidine ring brought about by alkyl substitution. It was found that each -CH2-group added with an alkyl substitution contributes favorably about -20 kJ mol(-1) toDeltaH degrees (int).This contribution is partially cancelled by the unfavorable contribution to DeltaH degrees (int) connected with removal of some water molecules bound in the first and subsequent hydration layers by an alkyl substituent. This is particularly evident on substitution at the polar side of the diketopyrimidine ring on which water molecules are expected to be bound specifically.     

Structural characteristics of an abnormal protein influencing its proteolytic susceptibility.

Structural properties of two similar beta-galactosidase fragments were investigated to determine how they influence the fragments' degradation rate in Escherichia coli. Both fragments resulting from a C-terminal nonsense mutation in lacZ, the CSH11 polypeptide and its 90 kDa degradative intermediate, exist predominantly as monomer subunits instead of in the tetrameric form characteristic of the native enzyme. However, both fragments appear to produce trace amounts of dimers and tetramers. The tetramer and higher molecular weight aggregates formed by the wild-type subunit confer greater protection for the enzyme's N-terminal auto-alpha polypeptide than does the monomer state of the beta-galactosidase fragments. The thermally induced aggregation of both beta-galactosidase fragments correlates with their sensitivity to alpha-chymotrypsin. The relatively low thermal stability of the 90 kDa degradative intermediate appears to be the cause of the significant increase in its proteolytic susceptibility at moderately high temperatures.