Molecular characterization of human and mouse fatty acid amide hydrolases

Recently, we reported the isolation, cloning, and expression of a rat enzyme, fatty acid amide hydrolase (FAAH), that degrades bioactive fatty acid amides like oleamide and anandamide to their corresponding acids, thereby serving to terminate the signaling functions of these molecules. Here, we report the molecular characterization of both a mouse and a human FAAH and compare these enzymes to the rat FAAH. The enzymes are well conserved in primary structure, with the mouse and rat FAAHs sharing 91% amino acid identity and the human FAAH sharing 82% and 84% identity with the rat FAAH and mouse FAAH, respectively. In addition, the expressed human and rat FAAHs behave biochemically as membrane proteins of comparable molecular size and show similar, but distinguishable, enzymological properties. The identification of highly homologous FAAH proteins in rat, mouse, and human supports a general role for the fatty acid amides in mammalian biology.     

A combination of a common splice site mutation and a frameshift mutation in the COL7A1 gene: absence of functional collagen VII in keratinocytes and skin

Musk xylene (2,4,6-trinitro-1-t-butylxylene; MX) is a synthetic nitromusk perfume ingredient that induces and inhibits mouse cytochrome P4502B (CYP2B) enzymes in vivo. The purpose of the present work was to determine whether amine metabolites of MX contributed to the enzyme inhibition and, if so, to define the nature and kinetics of this inhibition. When dosed orally to phenobarbital (PB)-treated mice, MX (200 mg/kg) inhibited > 90% of the PB-induced O-dealkylation of 7-pentoxyresorufin (PROD), and [14C]MX equivalents bound covalently to microsomal proteins. However, when this experiment was repeated in mice pretreated with antibiotics to eliminate the gastrointestinal flora, no decrease in PB-induced PROD activity and no covalent binding to microsomal proteins were observed. Thus, the ability of antibiotic treatment to eliminate the enzyme inhibition and covalent binding implicated amine metabolites of MX formed by nitroreduction in anaerobic intestinal flora as obligatory for these effects. Two monoamine metabolites of MX were synthesized to study enzyme inhibition directly. These metabolites were 2-amino-4,6-dinitro-1-t-butyl-xylene and 4-amino-2,6-dinitro-1-t-butylxylene, referred to as o-NH2-MX and p-NH2-MX, respectively, reflecting the position of the amine substitution relative to the t-butyl function. In the in vitro studies with PB-induced mouse liver microsomes, both amines inhibited PROD activity when preincubated in the absence of NADPH. However, only p-NH2-MX caused a time- and NADPH-dependent loss of PROD activity, and the inactivation rate was a pseudo-first-order process that displayed saturation kinetics. These results indicate that p-NH2-MX is a mechanism-based inactivator of mouse CYP2B enzymes. From kinetic analyses, the Ki was calculated to be 10.5 microM and the Kinact was 1.2 min-1. As final confirmation of the inhibitory effects of p-NH2-MX on mouse CYP2B enzymes, the amine (0.67 mmol/kg) was dosed orally to PB-induced mice. At 2 hr after dosing, p-NH2-MX inhibited essentially all of the PB-induced PROD activity, whereas an equimolar dosage of parent MX had no effect at this early time. Thus, although MX is an inducer of mouse CYP2B enzymes, an amine metabolite of MX is a mechanism-based inactivator of mouse CYP2B10. Furthermore, it is likely that the amine is responsible for the lack of functional CYP2B enzyme activity associated with induction of this enzyme by MX.     

Effects of dilute acetic acid on the cervical smear

BACKGROUND: Mesothelial integrity is essential for the prevention of pericardial adhesions. This study was performed to determine the effect of physical protection of the pericardium on mesothelial integrity. METHODS: A pericardial biopsy specimen was obtained at the time of pericardiotomy (0 minutes) in 10 patients undergoing a cardiac operation for the first time. The left free edge of the pericardiotomy was plicated inward to protect the mesothelium. Biopsy specimens were obtained from the protected and unprotected pericardium at 45 and 90 minutes after the start of extracorporeal circulation. Mesothelial integrity and the local inflammatory response were then assessed and graded histologically. RESULTS: The mesothelium was found to be present in the protected specimens at 0, 45, and 90 minutes, but it was found to be denuded in the unprotected specimens (p = 0.003 at 45 minutes; p = 0.004 at 90 minutes). Local inflammation was totally established in both the protected and unprotected specimens at 45 minutes. CONCLUSIONS: Physical agents appear to be the main factor that is damaging to the pericardial mesothelium, and this is an important concept to be taken into consideration when designing a method to prevent pericardial adhesions.     

Accumulation of pyrraline-modified albumin in phagocytes due to reduced degradation by lysosomal enzymes

Previous studies suggested that the interaction between proteins modified by advanced glycation end products (AGEs) and cells, such as macrophages, may be involved in diabetic angiopathy. Pyrraline is one of the AGEs and known to be elevated in plasma of diabetic rats and humans, and is present in vascular lesions of diabetic and elderly subjects. We examined whether modification of albumin by pyrraline influences its degradation by macrophage-like cell line, P388D1 cells. Degradation of pyrraline-modified albumin by these cells was diminished, causing accumulation of the albumin in these cells. The susceptibility of pyrraline-modified albumin to lysosomal proteolytic enzymes was reduced by approximately 40% in vitro, while lysosomal activity in the cells per se was not affected. This phenomenon was also observed when human monocytes were used instead of P388D1 cells. Our results suggest that accumulation of pyrraline-modified albumin in P388D1 cells is due to the reduced susceptibility of the protein to lysosomal enzymatic degradation. Such alterations in the interaction between AGEs-modified protein and phagocytes may contribute to angiopathy in elderly subjects and patients with diabetes.     

Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts

The proliferative capacity and cellular and biochemical characteristics of human trabecular bone osteoblasts were analysed throughout their replicative lifespan in vitro. Like several other cell types, human osteoblasts demonstrated a typical Hayflick phenomenon of cellular aging comprising a period of rapid proliferation until cumulative population doubling level (CPDL) 22 to 24, followed by a phase of slow growth and the final cessation of cell division at CPDL 32 to 34. Comparing young cells (less than 20% lifespan completed) and old cells (more than 90% lifespan completed) revealed a progressive increase in population doubling (PD) time, a decrease in attachment frequency, a decrease in the number of S-phase positive cells, a decrease in the rates of DNA, RNA and protein synthesis, an increase in the protein content per cell and an increased proportion of senescence-specific beta-galactosidase positive cells. While osteoblastic production of collagen type I decreased progressively during aging, alkaline phosphatase activity dropped rapidly after the first few passages and then remained constant during the rest of the proliferative lifespan, Significant morphological changes from thin and spindle-shaped early passage young cells to large, flattened and irregularly shaped late passage old cells full of intracellular debris were observed. In comparison, osteoblasts established from an osteoporotic bone sample showed a maximum CPDL of less than 5, had a longer PD time and exhibited abnormal senescent morphology. Thus, we have demonstrated for the first time that human osteoblasts, like several other diploid cell types, have a limited proliferative capacity in vitro and undergo aging and senescence as measured by various cellular and biochemical markers. In addition, preliminary studies show that cells from osteoporotic bone have a severely reduced proliferative capacity. This model of bone cell aging facilitates study of the molecular mechanisms of osteoblast senescence as well as factors related to osteoblast dysfunction in patients with osteoporosis.     

Toward targets for initiation of chronic dialysis

OBJECTIVES: To better define the targets for initiation of chronic dialysis, we compared the relationship between the normalized protein equivalent of nitrogen appearance (nPNA, g/kg standard weight/day) and weekly urea clearance (Kt) normalized to total body water (V) in predialysis chronic renal failure (CRF) patients and in patients on continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD). We also studied the relationships of other nutritional parameters to weekly Kt/Vurea in CRF patients. DESIGN: This cross-sectional study was a prospective observational design meant to study each patient once. SETTING: The University Hospital and Clinics and Harry S. Truman VA Medical Center, Columbia, Missouri. PATIENTS: Forty-five consecutive predialysis CRF patients were enrolled and the results compared with patients on CAPD and HD. RESULTS: In CRF, the nPNA calculated from urea appearance correlated with the weekly Kt/Vurea (r = 0.57, p < 0.0001) and, using exponential best-fit, nPNA = 1.217 x (1-e-0.769Kt/V). This exponential relationship was similar to that for CAPD and both were different from that in patients on HD. Likewise, nPNAs, calculated from Kjeldahl nitrogen output, and weekly Kt/Vurea were correlated (r = 0.37, p = 0.014) and, using exponential best-fit, nPNA = 1.102(1-e-0.867Kt/V), similar to the relationship in patients on CAPD. Evidence is presented that these relationships are not explained only by mathematical coupling. There was a significant correlation between the weekly Kt/Vurea and 24-hour urinary creatinine excretion. CONCLUSIONS: The findings suggest that in CRF, as in CAPD, a weekly Kt/Vurea less than 2.0 is likely to be associated with a nPNA less than 0.9 g/kg standard weight. In CRF patients, initiation of chronic dialysis should be considered if weekly renal Kt/Vurea falls below 2.0 and a nPNA greater than 0.8 is desired.     

Volatile anaesthetics reduce adhesion of blood platelets under low-flow conditions in the coronary system of isolated guinea pig hearts

BACKGROUND: Inhibitory effects of volatile anaesthetics on platelet aggregation have been demonstrated in several studies. However, the influence of volatile anaesthetics on intracoronary platelet adhesion has not been elucidated so far. METHODS: Isolated hearts of guinea pigs were perfused with buffer in the absence or presence of volatile anaesthetics (0.5 and 1 MAC) at constant coronary flow rates of 5 ml/min for 25 min, then 1 ml/min for 30 min and again 5 ml/min for 10 min. Before, during and after low-flow perfusion, a bolus of human platelets was applied into the coronary system. To simulate thrombogenic conditions, 0.3 U/ml human thrombin was infused during low-flow perfusion and reperfusion. The number of platelets sequestered to the endothelium was calculated from the difference between coronary in- and output of platelets. The myocardial production of lactate and consumption of pyruvate and coronary perfusion pressure were also determined. RESULTS: At a flow rate of 5 ml/min only about 3% of the applied platelets did not emerge from the coronary system, in any group. In contrast, 13.1 +/- 1.2% (mean +/- SEM) of infused platelets became adherent in low-flow perfusion in the control group without anaesthetic. The adherence was reduced with each 1 MAC isoflurane (to 6.2 +/- 1.2%), sevoflurane (to 4.4 +/- 0.9%) or halothane (to 3.2 +/- 1.5%) (each P < 0.05 vs. control). Volatile anaesthetic, 0.5 MAC, did not inhibit platelet adhesion to a statistically significant extent in any case. Perfusion pressure and metabolic parameters were not statistically different between the control and the hearts exposed to anaesthetics. CONCLUSION: Volatile anaesthetics in a concentration of 1 MAC can reduce the adhesion of platelets in the coronary system under reduced flow conditions. This action does not arise from vasodilation or inhibition of ischaemic stress.     

Autosomal dominant medullary cystic disease: a disorder with variable clinical pictures and exclusion of linkage with the NPH1 locus

BACKGROUND: The nephronophthisis-medullary cystic disease (NPH/MCD) complex represents a heterogeneous group of hereditary tubulointerstitial nephritis. The most common variant is juvenile recessive NPH, for which a gene locus (NPH1) has been mapped on chromosome 2q13. MCD is a less common dominant condition usually recognized later in life, which resembles NPH in many aspects, still presenting remarkable clinical differences. Nothing is known about the chromosome locus of MCD. METHODS: Five MCD families were studied. Diagnosis was made by inference from family history, type of inheritance, clinical signs and histology. Multipoint linkage analysis was performed by markers D2S293, D2S340 and D2S160 spanning the entire NPH1 locus. RESULTS: Diagnosis of MCD was made in 28 affected members (16 males; 12 females), belonging to five families. Histological diagnosis was available in 10 patients; clinical diagnosis in 11; seven deceased relatives had diagnosis of chronic nephritis. The age at diagnosis ranged from 8 to 65 years. Renal medullary cysts were found in a minority of patients. In family 1, the disease was associated with hyperuricaemia and gouty arthritis. Progression of renal disease presented intra- and extra-family variability with members of the same family showing mild elevation of creatinine or terminal renal failure. The NPH1 locus associated to recessive NPH was excluded from linkage to the dominant MCD. CONCLUSIONS: MCD might be more common than previously assumed. Variability in clinical presentation and absence of histopathological hallmarks contribute to make the diagnosis uncommon. The most remarkable clinical difference with NPH is the age of onset in some kindreds and a delayed progression towards renal failure. The exclusion of linkage to the NPH1 locus suggests the existence of an MCD responsible locus, still to be mapped.