SPLASH: systematic proteomics laboratory analysis and storage hub

In the field of proteomics, the increasing difficulty to unify the data format, due to the different platforms/instrumentation and laboratory documentation systems, greatly hinders experimental data verification, exchange, and comparison. Therefore, it is essential to establish standard formats for every necessary aspect of proteomics data. One of the recently published data models is the proteomics experiment data repository [Taylor, C. F., Paton, N. W., Garwood, K. L., Kirby, P. D. et al., Nat. Biotechnol. 2003, 21, 247-254]. Compliant with this format, we developed the systematic proteomics laboratory analysis and storage hub (SPLASH) database system as an informatics infrastructure to support proteomics studies. It consists of three modules and provides proteomics researchers a common platform to store, manage, search, analyze, and exchange their data. (i) Data maintenance includes experimental data entry and update, uploading of experimental results in batch mode, and data exchange in the original PEDRo format. (ii) The data search module provides several means to search the database, to view either the protein information or the differential expression display by clicking on a gel image. (iii) The data mining module contains tools that perform biochemical pathway, statistics-associated gene ontology, and other comparative analyses for all the sample sets to interpret its biological meaning. These features make SPLASH a practical and powerful tool for the proteomics community.     

Cell-free synthesis of rat liver zinc-thioneins.

The induction of rat liver zinc-thioneins mRNA was studied in a wheat germ cell-free translation system. Liver poly A rich polysomal RNA was isolated from rats which had been injected with zinc sulfate 5 h previously. These RNA preparations stimulated the incorporation of [35S]cystine into trichloroacetic acid insoluble proteins when assayed in the cell-free synthetic system. The translation products were characterized by Sephadex G-75 chromatography in 8 M urea--50 mM beta-mercaptoethanol, by disc gel electrophoresis in 4 M area--Tris-glycine buffer (pH 9.2), and by peptide fingerprinting with pepsin. These results were identical with authentic rat liver zinc-thioneins. The zinc-thioneins mRNA activity in the control rats, however, was minimal. The stimulation in zinc-thioneins synthesis observed in the cell-free synthesis was similar to the increased synthesis of these polypeptides in vivo.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Comparative proteomic analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli strains and their ihf and ler mutants

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.     

Crystal structures of major envelope proteins VP26 and VP28 from white spot syndrome virus shed light on their evolutionary relationship

White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 A, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt beta-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core beta-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.     

Evaluation of Buprenorphine in a Postoperative Pain Model in Rats

We evaluated the commonly prescribed analgesic buprenorphine in a postoperative pain model in rats, assessing acute postoperative pain relief, rebound hyperalgesia, and the long-term effects of postoperative opioid treatment on subsequent opioid exposure. Rats received surgery (paw incision under isoflurane anesthesia), sham surgery (anesthesia only), or neither and were treated postoperatively with 1 of several doses of subcutaneous buprenorphine. Pain sensitivity to noxious and nonnoxious mechanical stimuli at the site of injury (primary pain) was assessed at 1, 4, 24, and 72 h after surgery. Pain sensitivity at a site distal to the injury (secondary pain) was assessed at 24 and 72 h after surgery. Rats were tested for their sensitivity to the analgesic and locomotor effects of morphine 9 to 10 d after surgery. Buprenorphine at 0.05 mg/kg SC was determined to be the most effective; this dose induced isoalgesia during the acute postoperative period and the longest period of pain relief, and it did not induce long-term changes in opioid sensitivity in 2 functional measures of the opioid system. A lower dose of buprenorphine (0.01 mg/kg SC) did not meet the criterion for isoalgesia, and a higher dose (0.1 mg/kg SC) was less effective in pain relief at later recovery periods and induced a long-lasting opioid tolerance, indicating greater neural adaptations. These results support the use of 0.05 mg/kg SC buprenorphine as the upper dose limit for effective treatment of postoperative pain in rats and suggest that higher doses produce long-term effects on opioid sensitivity.Relief of postoperative pain is mandated in the Guide for the Care and Use of Animals¹⁸ and the Public Health Service Policy¹⁷ and is a major objective of laboratory animal medicine. Buprenorphine is one of the most commonly used opioid analgesics for postoperative pain in laboratory animals, mainly because of its long duration of action.¹⁰ The typical recommended dose range of buprenorphine in rats is 0.02 to 0.05 mg/kg SC.¹⁰ The upper end of this range, although effective at relieving acute postoperative pain in rats, is associated with side effects such as enhanced postoperative pain after the drug has worn off (rebound hyperalgesia),²³ respiratory depression,²¹ nausea or gastrointestinal distress and pica,²⁵ and neural adaptations (for example, sensitization) that may lead to long-term changes in neural function in the central nervous system and consequent changes in behavior.¹⁴ Central sensitization is a well-studied neural adaptation expressed in the brain and spinal cord and induced by nociceptive stimulation (that is, pain-induced by surgical manipulation) that manifests as hyperalgesia (decreased pain threshold to noxious stimuli) and allodynia (appearance of pain-like responses to nonnoxious tactile stimuli) during the recovery period.^16,29 Central sensitization contributes to persistent pain during the postoperative recovery period (that is, maintenance of increased pain sensitivity during tissue recovery) and chronic pain in some pathologic conditions (that is, persistent pain sensitivity after full tissue recovery). Central sensitization also accounts for the spread of hyperalgesia and allodynia to noninjured areas of the body distal to the injury.³¹ This phenomenon is referred to as ‘secondary pain’ (secondary hyperalgesia and allodynia), because it is not directly associated with the primary injury site.Opioid analgesics inhibit pain by acting on the nervous system to block transduction of pain signals traveling in sensory neurons toward the central nervous system and by facilitating activity of the descending pain inhibition neural pathway.¹⁶ Opioid analgesics also induce neural adaptations in the nervous system, phenomena that underlie the pronounced changes in behavior associated with addiction to narcotics.² Notably, opioid analgesics have been shown to enhance central sensitization initiated by pain transmission.^6,8,14,20 This property means that opiate analgesics facilitate both the inhibition of pain and central sensitization that leads to the enhancement of pain. Because central sensitization is a neural adaptation, the interaction of opiates on this pain mechanism outlasts the presence of the drug; in contrast, opiate effects on pain inhibition are limited to the presence of the drug. This arrangement is thought to account for rebound pain, that is, increased pain sensitivity after the opiate analgesic has worn off. Opiate side effects can compromise the success of recovery by increasing the level of distress experienced during recovery (for example, inducing nausea) and possibly increasing the duration of distress during recovery (for example, allowing for rebound pain). Moreover, and of importance specifically to laboratory animal medicine, the general neural adaptations induced by even a single dose of an opiate analgesic²⁶ may induce changes in the nervous system that alter and therefore compromise the validity of the animal model under study (for example, opioid mechanisms involved in behavioral control).We previously evaluated the feasibility of oral administration of buprenorphine.^15,25 As a basis for comparison, we used the ‘gold-standard’ postoperative buprenorphine dose of 0.05 mg/kg SC. The results of those studies showed that oral administration of buprenorphine was not feasible because the dose necessary to produce analgesia comparable to the standard dose of 0.05 mg/kg SC was 10 times the oral dose recommended in the literature and because the resulting concentration of oral buprenorphine was too bitter for rats to ingest voluntarily in a volume of flavored foodstuff that they could eat in a single meal.^15,25 We also observed that both subcutaneous and oral buprenorphine caused conditioned aversion to flavors,²⁵ suggestive of gastrointestinal distress⁵, with a greater effect for the oral route. Our conclusions and the associated clinical recommendation were limited by our presumption that buprenorphine at 0.05 mg/kg SC was the ideal postsurgical dose.An assessment of the literature that established this dose identified 2 problems. First, little or no research had directly assessed the effect of buprenorphine on pain sensitivity in animals in the hyperalgesic state that characterized the postoperative period,²³ and to our knowledge, no study has directly assessed the dose–response function of postsurgical buprenorphine on hyperalgesia. We hypothesized that endogenous opioids activated during the postoperative period²⁴ might act synergistically with buprenorphine to allow adequate relief of postoperative pain with a lower dose of buprenorphine than is necessary in an algesiometric test, thereby making predictions and extrapolations from algesiometric tests inaccurate. Second, we found that little consideration had been given to the consequences of other physiologic effects of buprenorphine on the recovery process (for example, gastrointestinal distress⁵, rebound hyperalgesia, and allodynia). As stated earlier, recent research on central sensitization has determined that although opioid analgesics inhibit pain sensation acutely, they also enhance neural adaptations that account for rebound pain and other long-term chronic pain conditions.^16,28,29,31 We hypothesized secondarily that a lower dose of buprenorphine, if effective acutely, would result in reduced side effects and be less likely to initiate or enhance neural adaptations, such as rebound hyperalgesia and allodynia.The current study had 2 goals. The first was to establish the minimum dose of buprenorphine needed to relieve acute postoperative pain effectively in rats. As a starting point, we defined effective relief of acute pain as the induction of isoalgesia during the postoperative period; isoalgesia is the normal level of pain sensation, in contrast to analgesia (absence of pain sensation) or hypoalgesia (lower-than-normal pain sensation). The second goal was to evaluate the effect of postoperative buprenorphine on factors that slow recovery (that is, rebound hyperalgesia and allodynia) or create long-term changes (that is, sensitization or tolerance to opiates). We tested our hypothesis by using various doses of buprenorphine in a rat model of incisional pain.^3,4,31 This model was selected because it induces cutaneous and muscular pain common to most surgery and generates mild to moderate persistent pain so that both the acute inhibitory effects of the buprenorphine (that is, pain relief) and the lasting effects of buprenorphine (that is, rebound hyperalgesia) could be studied.