Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid that activates cells involved in inflammation. The biological activity of PAF depends on its structural features, namely an ether linkage at the sn-1 position and an acetate group at the sn-2 position. The actions of PAF are abolished by hydrolysis of the acetyl residue, a reaction catalysed by PAF acetylhydrolase. There are at least two forms of this enzyme--one intracellular and another that circulates in plasma and is likely to regulate inflammation. Here we report the molecular cloning and characterization of the human plasma PAF acetylhydrolase. The unique sequence contains a Gly-Xaa-Ser-Xaa-Gly motif commonly found in lipases. Recombinant PAF acetylhydrolase has the substrate specificity and lipoprotein association of the native enzyme, and blocks inflammation in vivo: it markedly decreases vascular leakage in pleurisy and paw oedema, suggesting that PAF acetylhydrolase might be a useful therapy for severe acute inflammation.

Neuromedin U (NMU) is a highly conserved neuropeptide with a variety of physiological functions mediated by two receptors, peripheral NMUR1 and central nervous system NMUR2. Here we report the generation and phenotypic characterization of mice deficient in the central nervous system receptor NMUR2. We show that behavioral effects, such as suppression of food intake, enhanced pain response, and excessive grooming induced by intracerebroventricular NMU administration were abolished in the NMUR2 knockout (KO) mice, establishing a causal role for NMUR2 in mediating NMU's central effects on these behaviors. In contrast to the NMU peptide-deficient mice, NMUR2 KO mice appeared normal with regard to stress, anxiety, body weight regulation, and food consumption. However, the NMUR2 KO mice showed reduced pain sensitivity in both the hot plate and formalin tests. Furthermore, facilitated excitatory synaptic transmission in spinal dorsal horn neurons, a mechanism by which NMU stimulates pain, did not occur in NMUR2 KO mice. These results provide significant insights into a functional dissection of the differential contribution of peripherally or centrally acting NMU system. They suggest that NMUR2 plays a more significant role in central pain processing than other brain functions including stress/anxiety and regulation of feeding.Neuromedin U (NMU) is a highly conserved neuropeptide, present in various species from amphibians to mammals (reviewed in reference 3). In humans, NMU is a 25-amino-acid (aa) peptide (NMU-25), and in rodents, it is a 23-aa peptide (NMU-23), whereas in some other mammalian species an 8-aa peptide (NMU-8) has also been found. NMU-8 is identical to the C terminus of NMU-25, which is the most highly conserved region of the entire peptide, and has receptor affinity in vitro similar to that of NMU-25. NMU is widely distributed in the body, with the most abundant expression in the gastrointestinal tract, anterior pituitary, spinal cord, brain, and genitourinary tract (6, 42). Correspondingly, NMU has been implicated in regulating a variety of physiological functions, including smooth-muscle contraction, blood pressure regulation, stress response, feeding and energy homeostasis, nociception, and circadian rhythm (reviewed in reference 3).Two G-protein-coupled receptors, NMUR1 and NMUR2, have been identified as the receptors for NMU (8,18,20,21,37,41,42). The two receptors belong to the rhodopsin-like class A G-protein-coupled receptors family and share ϳ50% identity with each other in the seven-transmembrane region. The tissue distribution of the two receptors is quite distinct and complementary to each other: NMUR1 is expressed predominantly in the periphery, with highest levels in the gastrointestinal tract (8,10,18,42), whereas NMUR2 is predominantly expressed in the central nervous system, with greatest expression in regions of hypothalamus, medulla, and spinal cord (9,10,14,20,21,41).In the brain, NMU is expressed in hypothalamic regions associated with regulation of food intake and energy homeostasi...

Rats within the early maintenance phase of post-ischemic acute renal failure (ARF) can resist additional ischemic insults. This study assessed whether this protection exists directly at the tubular cell level, and if so, whether it is a consequence of prior cell injury (for example, due to heat-shock protein synthesis; HSP), or if it arises in response to reductions in functional renal mass and/or the uremic environment. Rats were subjected to either 15 or 35 minutes of unilateral or bilateral renal ischemia, and after 15 minutes to 24 hours of reflow, proximal tubular segments (PTS) were isolated for study. Their viability following oxygenation and hypoxic/reoxygenation injury (H/R) was tested (LDH release). The influence of uremia/reduced renal mass was determined by studying PTS extracted 24 hours after 1 1/2 nephrectomy, and by determining whether PTS exposure to a "uremic milieu" (urine addition) blocks H/R damage. HSP effects were gauged by correlating renal cortical HSP-70 expression with degrees of in vitro protection, and by ascertaining whether in vivo hyperthermia (42 degrees C; 15 min) mitigates subsequent PTS H/R damage. Results were compared with those obtained from normal PTS. The in vivo experimental protocols did not substantially alter PTS isolation or their viability during oxygenation. Fifteen minutes of ischemia induced neither azotemia nor PTS cytoprotection. In contrast, 35 minutes of ischemia conferred marked protection against subsequent H/R, but only when azotemia was permitted to develop (protection seen after 24 hr, but not at 4 hr of reflow; protection abrogated by retention of 1 normal kidney). Renal failure in the absence of tubular necrosis (1 1/2 uninephrectomy) protected PTS from H/R damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.

TRH is a neuropeptide with a variety of hormonal and neurotransmitter/neuromodulator functions. In particular, TRH has pronounced acute antidepressant effects in both humans and animals and has been implicated in the mediation of the effects of other antidepressive therapies. Two G protein-coupled receptors, TRH receptor 1 (TRH-R1) and TRH-R2, have been identified. Here we report the generation and phenotypic characterization of mice deficient in TRH-R1. The TRH-R1 knockout mice have central hypothyroidism and mild hyperglycemia but exhibit normal growth and development and normal body weight and food intake. Behaviorally, the TRH-R1 knockout mice display increased anxiety and depression levels while behaving normally in a number of other aspects examined. These results provide the first direct evidence that the endogenous TRH system is involved in mood regulation, and this function is carried out through TRH-R1-mediated neural pathways.

After O2 deprivation, tissue acidosis rapidly self-corrects. This study assessed the effect of this pH correction on the induction, and pathways, of posthypoxic proximal tubular injury. In addition, ways to prevent the resultant injury were explored. Isolated rat proximal tubular segments (PTSs) were subjected to hypoxia/reoxygenation (50/30 or 30/50 minutes) under the following incubation conditions: 1) continuous pH 7.4, 2) continuous pH 6.8, or 3) hypoxia at pH 6.8 and reoxygenation at pH 7.4 (NaHCO3 or Tris base addition). Continuously oxygenated PTSs maintained under these same pH conditions served as controls. Lethal cell injury was assessed by lactate dehydrogenase (LDH) release. pH effects on several purported pathways of hypoxia/reoxygenation injury were also assessed (ATP depletion, lipid peroxidation, and membrane deacylation). Acidosis blocked hypoxic LDH release (pH 7.4, 50 +/- 2%; pH 6.8, 6 +/- 1%) without mitigating membrane deacylation or ATP depletion. During reoxygenation, minimal LDH was released (3-5%) if pH was held constant. However, if posthypoxic pH was corrected, immediate (< or = 5 minutes) and marked cell death (e.g., 55 +/- 3% with Tris) occurred. This was dissociated from lipid peroxidation or new deacylation, and it was preceded by a depressed ATP/ADP ratio (suggesting an acidosis-associated defect in hypoxic/posthypoxic cell energetics). Realkalinization injury was not inevitable, since it could be substantially blocked by 1) posthypoxic glycine addition, 2) transient posthypoxic hypothermia, or 3) allowing a 10-minute reoxygenation (cell recovery) period before base addition. Neither mannitol nor graded buffer Ca2+ deletion conferred protection. Acute pH correction caused no injury to continuously oxygenated PTSs. Conclusions are as follows: 1) Posthypoxic "pH shock" causes virtually immediate cell death, not by causing de novo injury but, rather, by removing the cytoprotective effect of acidosis. 2) This injury can be prevented by a variety of methods, indicating a great potential for salvaging severely damaged posthypoxic PTSs.

No abstract

Amphotericin B (AB) may induce acute renal failure by vasoconstrictive and tubulo-toxic effects. Although mannitol, Ca2+ channel blockers, and lipid-based AB preparations have been suggested to mitigate in vivo AB nephrotoxicity, whether they confer direct tubular cytoprotection has not been defined. Therefore, this study assessed the impact of mannitol, verapamil/extracellular Ca2+, and cholesteryl sulfate (CS) AB binding on AB cytotoxicity, employing an isolated rat proximal tubular segment (PTS) preparation. After 30 to 60 minutes of incubation, 0.2 mg/ml of AB (Fungizone) caused marked toxicity, as assessed by LDH release (29 to 44%) and ATP depletion (greater than 90%). Approximately 40% of the LDH release could be attributed to deoxycholate, the standard AB (Fungizone) solubilizing agent. Both 100 mM mannitol and 100 mM glucose decreased AB-mediated LDH release, despite having a quantitatively trivial impact on ATP concentrations (increments of less than or equal to 1% at normal values). Dimethylthiourea (25 mM; equipotent to 100 mM mannitol/glucose as a hydroxyl radical scavenger) did not decrease LDH release. Neither verapamil addition (100 microM) nor Ca2+ removal from the PTS buffer had a protective effect. CS binding completely eliminated AB's toxicity (no LDH or ATP losses). The effect of AB and CS-AB on concomitant O2 deprivation/reoxygenation (30 min/15 min) PTS injury was also assessed. AB and hypoxia/reoxygenation caused additive, not synergistic, LDH release whereas CS-AB had no adverse effect.(ABSTRACT TRUNCATED AT 250 WORDS)