The maturation of the neuromodulatory action of substance P (SP) was investigated in tracheal smooth muscle (TSM) segments isolated from rabbits aged 2-24 wk. The tissues were placed in baths containing Krebs-Ringer solution and contracted with electrical field stimulation (ES) with ES frequencies ranging from 1 to 75 Hz. In tissues greater than 1 mo of age, the ES frequency-response relationships were progressively shifted in the presence of a maximally effective neuromodulatory SP dose (10(-7) M) such that by 24 wk of age the mean (+/- SEM) maximal tension (Tmax) significantly increased from 380.4 (+/- 41.9) to 502.3 (+/- 64.2) g/g TSM, and the corresponding mean (+/- SEM) log ES frequency producing 50% of Tmax (log ES50) significantly decreased from 1.209 (+/- 0.069) to 1.055 (+/- 0.046) Hz. By contrast, relative to methacholine, the direct contractile effects of SP did not significantly vary with age. In further analyzing the basis for the above age-related difference in the neuromodulatory action of SP, we found that the magnitude of SP-induced neuromodulation was highly correlated to the tissue's intrinsic sensitivity to ES. Indeed, after accounting for the tissue's sensitivity to ES, the effect of age alone on the magnitude of SP-induced neuromodulation was not statistically significant. These findings provide new evidence that: (a) SP-induced neuromodulation of acetylcholine release at the airway neuromuscular junction is significantly enhanced during postnatal development; and (b) that the latter age-dependent action of SP is based on a close coupling of the magnitude of SP-induced neuromodulation to the tissue's intrinsic sensitivity to neurally mediated contraction.
D T Tanaka, M M Grunstein
Attachment of pathogens to host cells is a prerequisite for the development of many infections. Pneumocystis carinii (PC) pneumonia is characterized by attachment of PC trophozoites to the alveolar epithelium. The mechanism of this process is unknown. Fibronectin (Fn) is a glycoprotein present in the alveolar space known to mediate cell-cell attachment, including the attachment of certain pathogens to host epithelial cells. In this study the binding of Fn to PC trophozoites has been characterized in vitro using 125I-Fn. Fn binds saturably and specifically to 6.4 x 10(5) binding sites per organism with an apparent binding constant, Kd, of 1.2 x 10(-8) M. Fn binding to PC was inhibited by the addition of Arg-Gly-Asp-Ser (RGDS), a tetrapeptide containing the active site of the cell-binding domain of Fn. PC attachment to an alveolar epithelial cell line was quantified using 51Cr-labeled PC trophozoites. Attachment was decreased from 24 +/- 1.9% to 12.1 +/- 1% (P less than 0.01) by the addition of an anti-Fn antibody, an effect that could be overcome by the addition of excess free Fn. It is concluded that binding of Fn to PC may be an important initial step in the attachment of the organism to alveolar epithelial cells. Furthermore, it appears that PC recognizes and binds to the RGDS cell attachment site of Fn.
S T Pottratz, W J Martin 2nd
Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.
E M Quinet, L B Agellon, P A Kroon, Y L Marcel, Y C Lee, M E Whitlock, A R Tall
There are at least three major African haplotype backgrounds on which the beta s mutation arises. Sequence changes in the immediate 5' flanking area of the gamma-globin genes may account for differences in fetal hemoglobin expression among the three haplotypes. We determined the sequence from -350 to 10 bp 5' of the G gamma and A gamma fetal globin genes from one beta s-containing chromosome on each of the three major haplotype backgrounds. The Senegal chromosome had a T at -158 5' to the G gamma gene; the Benin (BEN) chromosome had an A to G change at -309 5' to the G gamma gene; and the Central African Republic (CAR) chromosome had a C to T change at -271 5' to the A gamma gene. Genomic DNA from patients with sickle cell disease was analyzed using the polymerase chain reaction and radiolabeled allele-specific oligonucleotide probes. The -309 G variant 5' to the G gamma gene is associated with BEN chromosomes, and the -271 T variant 5' to A gamma with CAR. The -309 change was also found on beta A-containing chromosomes, while the -271 change was not. The -309 change may have predated the beta s mutation on the BEN chromosome.
S R Month, R W Wood, P T Trifillis, P J Orchowski, B Sharon, S K Ballas, S Surrey, E Schwartz
Toxoplasma gondii is a common protozoan disease that often causes life-threatening disease, particularly among patients with the acquired immunodeficiency syndrome. This study demonstrates that the dihydropteroate synthase in T. gondii is kinetically distinct from the enzyme characterized from other sources and can be highly purified with a high yield using sequential dye-affinity chromatography. Conditions have been identified that allow for stabilization of the purified enzyme, and its physical characteristics have been elucidated. The molecular weight of the native protein was 125,000 and the protein appeared to contain both dihydropteroate synthase and 6-hydroxymethyl-dihydropterin pyrophosphokinase activities. The sulfonamide class of compounds vary in inhibitory potency by more than three orders of magnitude. Sulfathiazole, sulfamethoxazole, and sulfamethazine, with 50% inhibitory concentrations (IC50's) of 1.7, 2.7, and 5.7 microM, respectively, represent the most potent of this class of inhibitors. Several sulfone analogues, including dapsone, were identified as highly potent inhibitors with IC50's less than 1 microM. The results of these cell-free experiments were corroborated by investigating the metabolic inhibition produced by the various inhibitors in intact organisms. The qualitative and quantitative relations among the inhibitors were preserved in both the cell-free and intact cell assay systems. These studies suggest that the sulfones may be important therapeutic agents for the treatment of toxoplasmosis.
C J Allegra, D Boarman, J A Kovacs, P Morrison, J Beaver, B A Chabner, H Masur
Structure elucidation of a specific fluorophore from the aging extracellular matrix revealed the presence of a protein crosslink formed through nonenzymatic glycosylation of lysine and arginine residues. The unexpected finding that a pentose instead of a hexose is involved in the crosslinking process suggested that the crosslink, named pentosidine, might provide insight into abnormalities of pentose metabolism in aging and disease. This hypothesis was investigated by quantitating pentosidine in hydrolysates of 103 human skin specimens obtained randomly at autopsy. Pentosidine level was found to increase exponentially from 5 to 75 pmol/mg collagen over lifespan (r = 0.86, P less than 0.001). A three- to tenfold increase was noted in insulin-dependent diabetic and nondiabetic subjects with severe end-stage renal disease requiring hemodialysis (P less than 0.001). Moderately elevated levels were also noted in some very old subjects, some subjects with non-insulin dependent diabetes, and two subjects with cystic fibrosis and diabetes. The cause of the abnormal pentose metabolism in these conditions is unknown but may relate to hemolysis, impaired pentose excretion, cellular stress, and accelerated breakdown of ribonucleotides. Thus, pentosidine emerges as a useful tool for assessment of previously unrecognized disorders of pentose metabolism in aging and disease. Its presence in red blood cells and plasma proteins suggests that it might be used as a measure of integrated pentosemia in analogy to glycohemoglobin for the assessment of cumulative glycemia.
D R Sell, V M Monnier
Ethanol consumption retards the hepatic regenerative response to injury. This may contribute to the pathogenesis of liver injury in alcoholic individuals. The mechanisms responsible for ethanol-associated inhibition of liver regeneration are poorly understood. To determine if the antiregenerative effects of ethanol involve modulation of polyamine metabolism, parameters of polyamine synthesis were compared before and during surgically induced liver regeneration in ethanol-fed rats and isocalorically maintained controls. After partial hepatectomy, induction of the activity of ornithine decarboxylase (ODC), the rate limiting enzyme for polyamine synthesis, was delayed in rats that had been fed ethanol. This was correlated with reduced levels of putrescine, ODC's immediate product. Increases in hepatic spermidine and spermine were also inhibited. Differences in ODC activity between ethanol-fed and control rats could not be explained by differences in the expression of ODC mRNA or by differences in ODC apoenzyme concentrations, suggesting that chronic ethanol intake inactivates ODC posttranslationally. Supplemental putrescine, administered at partial hepatectomy and 4 and 8 h thereafter, increased hepatic putrescine concentrations and markedly improved DNA synthesis and liver regeneration in ethanol-fed rats. These data suggest that altered polyamine metabolism may contribute to the inhibition of liver regeneration that occurs after chronic exposure to ethanol.
A M Diehl, M Wells, N D Brown, S S Thorgeirsson, C J Steer
Pneumocystis carinii pneumonia is a significant cause of mortality in immunocompromised patients. Current concepts suggest that attachment of P. carinii to alveolar epithelium is required for development of pneumonia. We examined the mechanism of P. carinii adherence to cultured A549 cells, a permanent cell line derived from human alveolar epithelium. P. carinii adherence was quantified by measuring attachment of 51Cr-labeled P. carinii to cultured A549 cells. After 8 h of incubation, 37.4 +/- 4.2% of P. carinii were adherent to A549 cells. In the presence of agents known to impair cytoskeletal function, including 10(-5) M cytochalasin B, 10(-5) M colchicine, and 10(-5) M trimethylcolchicinic acid (TMCA), adherence was decreased from 57.4 +/- 4.2% to 9.3 +/- 3.4%, 12.5 +/- 3.6%, and 21.5 +/- 3.6%, respectively (P less than 0.01, all comparisons). Secondly, we examined the effect of P. carinii on the function of A549 cells. P. carinii resulted in significant impairment of A549 cell growth, indicating P. carinii adversely affected the function of target lung cells. A P. carinii:A549 cell ratio of 50:1 resulted in 43.5 +/- 2.9% inhibition of A549 cell growth (P less than 0.001). Additionally, TMCA, which significantly prevented attachment of P. carinii, reversed the impairment of A549 cell growth. These data demonstrate that P. carinii attachment to cultured lung cells can be quantified, is dependent on intact cytoskeletal function and is necessary for impairment of lung cell replication.
A H Limper, W J Martin 2nd
Plasma and lipoprotein alpha-tocopherol concentrations of four patients with familial isolated vitamin E deficiency and six control subjects were observed for 4 d after an oral dose (approximately 15 mg) of RRR-alpha-tocopheryl acetate labeled with six deuterium atoms (d6-tocopherol). Chylomicron d6-tocopherol concentrations were similar in the two groups. d6-Tocopherol concentrations of plasma, very low (VLDL), low (LDL), and high (HDL) density lipoproteins were similar in the two groups only during the first 12 h; then these were significantly lower, and the rate of disappearance faster, in the patients. The times (tmax) of the maximum chylomicron d6-tocopherol concentrations were similar for the two groups, but tmax values in the controls increased in the order: chylomicrons less than VLDL less than or equal to LDL approximately HDL, while the corresponding values in the patients were similar to the chylomicron tmax. Thus, plasma d6-tocopherol in controls increased during chylomicron and VLDL catabolism, whereas in patients it increased only during chylomicron catabolism, thereby resulting in a premature and faster decline in the plasma tocopherol concentration due to a lack of d6-tocopherol secretion from the liver. We suggest that these patients are lacking or have a defective liver "tocopherol binding protein" that incorporates alpha-tocopherol into nascent VLDL.
M G Traber, R J Sokol, G W Burton, K U Ingold, A M Papas, J E Huffaker, H J Kayden
Tetranectin, a protein recently identified in a wide variety of human secretory cells (Christensen, L., and I. Clemmensen. 1989. Histochemistry. 92:29-35) was found to colocalize with latent alkaline phosphatase activity in fractions well separated from azurophil granules, specific granules, gelatinase-containing granules, and plasma membranes when postnuclear supernatants of nitrogen-cavitated neutrophils were fractionated on discontinuous Percoll density gradients. Stimulation of intact neutrophils with nanomolar concentrations of FMLP, leukotriene B4, 10-100 U/ml of tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor resulted in parallel release of tetranectin and translocation of alkaline phosphatase to the plasma membrane. Furthermore, intracellular pools of tetranectin and latent alkaline phosphatase were completely released from neutrophils under conditions that barely induced release of specific granules containing B12-binding protein. These findings indicate that tetranectin and latent alkaline phosphatase define an easily mobilizable population of cytoplasmic storage organelles in human neutrophils which are functionally distinguishable from azurophil, specific, and gelatinase-containing granules. These organelles may play an important role as stores of membrane proteins that are mobilized to the cell surface during stimulation by inflammatory mediators.
N Borregaard, L Christensen, O W Bejerrum, H S Birgens, I Clemmensen
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