Evidence for an ancient chromosomal duplication in Arabidopsis thaliana by sequencing and analyzing a 400-kb contig at the APETALA2 locus on chromosome 4

As part of the European Scientists Sequencing Arabidopsis program, a contiguous region (396607 bp) located on chromosome 4 around the APETALA2 gene was sequenced. Analysis of the sequence and comparison to public databases predicts 103 genes in this area, which represents a gene density of one gene per 3.85 kb. Almost half of the genes show no significant homology to known database entries. In addition, the first 45 kb of the contig, which covers 11 genes, is similar to a region on chromosome 2, as far as coding sequences are concerned. This observation indicates that ancient duplications of large pieces of DNA have occurred in Arabidopsis.     

Mitochondria at the Crossroad of Apoptotic Cell Death

In the past few years, it has become widely appreciated that apoptotic cell death generallyinvolves activation of a family of proteases, the caspases, which undermine the integrity ofthe cell by cleavage of critical intracellular substrates. Caspases, which are synthesized asinactive zymogens, are themselves caspase substrates and this cleavage leads to their activation.Hence, the potential exists for cascades of caspases leading to cell death. However, it has beenrecently recognized that another, perhaps more prominent route to caspase activation, involvesthe mitochondria. Upon receipt of apoptotic stimuli, either externally or internally generated,cells initiate signaling pathways which converge upon the mitochondria to promote release ofcytochrome C to the cytoplasm; cytochrome c, thus released, acts as a potent cofactor incaspase activation. Even cell surface death receptors such as Fas, which can trigger directcaspase activation (and potentially a caspase cascade), appear to utilize mitochondria as partof an amplification mechanism; it has been recently demonstrated that activated caspases cancleave key substrates to trigger mitochondrial release of cytochrome c, thereby inducing furthercaspase activation and amplifying the apoptotic signal. Therefore, mitochondria play a centralrole in apoptotic cell death, serving as a repository for cytochrome c.     

The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70

The Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK. ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis. However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis. The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide. In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide. However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides. Binding of a fusion protein that contains a defective J-domain is insensitive to ATP. According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C). The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range. In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture. The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity. The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.     

Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.     

Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in gram-negative sepsis and causes inflammation and death in mice

Gram-negative bacterial sepsis commonly causes organ dysfunction and death in humans. Although circulating bacterial toxins trigger inflammation in sepsis, little is known about the composition of bacterial products released into the blood during sepsis or the contribution of various bacterial components to the pathogenesis of sepsis. We have shown that diverse Gram-negative bacteria release bacterial peptidoglycan-associated lipoprotein (PAL) into serum. The present studies explored release of PAL into the blood during sepsis and tested the hypothesis that PAL contributes to bacterial virulence and inflammation in Gram-negative sepsis. Released PAL was detected in the blood of 94% of mice following cecal ligation and puncture. Picomolar to nanomolar levels of PAL stimulated macrophages and splenocytes from lipopolysaccharide-hyporesponsive (C3H/HeJ) mice. Injection of PAL into C3H/HeJ mice stimulated production of serum cytokines and increased pulmonary and myocardial expression of inflammatory markers. PAL caused death in sensitized C3H/HeJ mice. Mutant Escherichia coli bacteria with reduced levels of PAL or truncated PAL were less virulent than wild-type bacteria, as indicated by higher survival rates and lower circulating levels of interleukin 6 and bacteria in a model of peritonitis in lipopolysaccharide-responsive mice. The studies suggest that PAL may be an important bacterial mediator of Gram-negative sepsis.     

Improvement in burn wound infection and survival with antimicrobial peptide D2A21 (Demegel)

Naturally occurring antimicrobial peptides have been discovered in both plants and animals. Many of these peptides demonstrate impaired activity or cytotoxicity when applied exogenously. Synthetically engineered antimicrobial peptides have been designed to increase potency and activity against bacteria and fungus yet remain noncytotoxic. The antimicrobial peptide D2A21 (Demegel) has already demonstrated significant activity in vitro against many common hospital pathogens. The purpose of this study was to evaluate the effects of D2A21 in an in vivo infected burn-wound model, examining both quantitative cultures of the wound and survival of the animal. Forty-four Wistar rats were subjected to a 23 percent total body surface area scald burn. Pseudomonas aeruginosa was administered topically with 108 organisms and wounds were then evaluated at day 1, 2, or 3 for eschar and subeschar muscle quantitative culture. The experimental group was treated daily with 1.5% topical D2A21. The control group was treated with control gel. A second group of Wistar rats (n = 14) were burned and given a 107 inoculum of the same Pseudomonas and evaluated to 14 days for survival and weight changes. This group was subdivided into rats receiving either topical D2A21 or control base daily. The quantitative biopsy results demonstrated that D2A21-treated wounds had no bacterial growth in burn eschar at day 2 or 3, whereas control animals demonstrated growth at greater than 105 organisms by day 2. Subeschar muscle cultures also demonstrated significantly less bacterial invasion compared with controls on each day tested. D2A21-treated animals had an 85.7 percent survival compared with 0 percent survival in controls. Furthermore, the D2A21-treated groups demonstrated maintenance of body weights, whereas controls had significant weight loss with time. In conclusion, D2A21 demonstrates significant antibacterial activity against Pseudomonas, sterilizing burn eschar and decreasing subeschar bacterial load, allowing for a markedly significant improvement in survival in this infected burn-wound model.     

Exploring human interaction and diet effects on the behavior of dogs in a public animal shelter

This study examined the effects of 2 manipulations-a brief, regular period of human contact and diet-on the behavior of dogs confined in a public animal shelter. A behavioral battery designed to assess reactions to novel situations, and a test of responsiveness to an unfamiliar human were administered both prior to (pretest) and immediately following (posttest) the 8-week intervention period. Overall, the regular periods of increased human contact together with a diet that contained augmented levels of digestible protein, fat, calories, and animal-derived ingredients reduced signs of behavioral reactivity from pretest to posttest. In some cases, the comparison diet appeared more effective, but only for dogs receiving minimal human interaction. The results indicate that a combination of human interaction and high quality diet may positively affect the behavior of dogs in animal shelters.     

ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities

ParA and ParB of Caulobacter crescentus belong to a conserved family of bacterial proteins implicated in chromosome segregation. ParB binds to DNA sequences adjacent to the origin of replication and localizes to opposite cell poles shortly following the initiation of DNA replication. ParA has homology to a conserved and widespread family of ATPases. Here, we show that ParB regulates the ParA ATPase activity by promoting nucleotide exchange in a fashion reminiscent of the exchange factors of eukaryotic G proteins. Furthermore, we demonstrate that ADP-bound ParA binds single-stranded DNA, whereas the ATP-bound form dissociates ParB from its DNA binding sites. Increasing the fraction of ParA-ADP in the cell inhibits cell division, suggesting that this simple nucleotide switch may regulate cytokinesis.     

Studies of Pseudomonas aeruginosa azurin mutants: cavities in beta-barrel do not affect refolding speed

Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold. Removal of copper produces an apoprotein with the same structure as holoazurin. To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a beta-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins. The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins. The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect. Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin. In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin. We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.     

Continuous protein recovery from whey using liquid-solid circulating fluidized bed ion-exchange extraction

A liquid-solid circulating fluidized bed (LSCFB) continuous ion-exchange extraction system has been investigated for total protein recovery from whey solutions under various operating conditions. The effectiveness of a dynamic seal was evaluated between the riser and the downcomer, and the best conditions for the establishment of this seal were established. Start-up studies indicated that the system is robust and stable. Under optimal conditions, a productivity of 8.2 g of total protein removed per hour per kilogram of resin was achieved with a protein removal efficiency of 78.4%. However, higher overall protein recovery of up to 90% was also achieved under other conditions, with lower protein concentration in the effluent and a lower overall productivity.