Abnormalities in lung surfactant in horses clinically affected with recurrent airway obstruction (RAO).

BACKGROUND: Abnormalities in lung surfactant are well described in human respiratory diseases including asthma, but are poorly described in horses. HYPOTHESIS: Lung surfactant is abnormal in horses with clinical signs of recurrent airway obstruction (RAO). ANIMALS: Six healthy horses and 5 horses with RAO. METHODS: Bronchoalveolar lavage fluid (BALF) was obtained from all horses by standard procedures. Cell-free BALF was separated into crude surfactant pellets (CSP) and supernatant via ultracentrifugation. Phospholipid and protein content was analyzed from both of these fractions. Phospholipid composition of CSP was determined using high-performance liquid chromatography with an evaporative light scatter detector. Surface tension of CSP was measured with a pulsating bubble surfactometer. RESULTS: Compared with healthy horses, surfactant from RAO-affected horses was characterized by significantly decreased phospholipid content in total surfactant (median; range: 23.2; 14.7-62.2 microg/mL BALF versus 172; 111-267 microg/mL BALF, P = .0062) and CSP (20.2; 6.4-48.9 microL/mL BALF versus 155; 94.4-248 microg/mL BALF, P = .0062), and a significantly lower percentage of phosphatidylglycerol (PG) (4.5; 3.6-5.6% versus 6.6; 4.1-7.6%, P = .028). Furthermore, the ratio between the percentages of phosphatidylcholine and PG was significantly higher in RAO-affected horses than in healthy horses (20.9; 16.6: 25.9 versus 13.9; 11.8-22.8, P = .045). CONCLUSIONS AND CLINICAL IMPORTANCE: This study demonstrates that surfactant from RAO-affected horses is abnormal. Further studies are needed to determine if these abnormalities are related to an increased tendency for bronchoconstriction and to a decreased ability to clear airway mucus in RAO-affected horses.

A burning love for lesbian literature.

ABSTRACT Barbara Grier's enjoyment of and commitment to lesbian literature have been guiding forces in her life. While working non-professional jobs for little pay, she managed to find money to buy the books she loved. Her enthusiasm led her to devote considerable time and energy to The Ladder while simultaneously working a full-time job. When The Ladder was no longer financially viable, she founded Naiad Press, and, for the first nine years of its existence, continued to hold a full-time job. In 1982, she became the first paid employee of Naiad, which enabled her to focus exclusively on her passions: writing, editing, and publishing lesbian literature.

The chondrodystrophy, nanomelia: biosynthesis and processing of the defective aggrecan precursor.

The lethal chicken mutation nanomelia leads to severe skeletal defects because of a deficiency of aggrecan, which is the largest aggregating chondroitin sulphate proteoglycan of cartilage. In previous work, we have demonstrated that nanomelic chondrocytes produce a truncated aggrecan precursor that fails to be secreted, and is apparently arrested in the endoplasmic reticulum (ER). In this study, we investigated the biosynthesis and extent of processing of the abnormal aggrecan precursor. The truncated precursor was translated directly in cell-free reactions, indicating that it does not arise post-translationally. Further studies addressed the processing capabilities of the defective precursor. We found that the mutant precursor was modified by N-linked, mannose-rich oligosaccharides and by the addition of xylose, but was not further processed; this is consistent with the conclusion that it moves no further along the secretory pathway than the ER. Using brefeldin A we demonstrated that the defective precursor can function as a substrate for Golgi-mediated glycosaminoglycan chains, but does not do so in the nanomelic chondrocyte because it fails to be translocated to the appropriate membrane compartment. These studies illustrate how combined cell biological/biochemical and molecular investigations may contribute to our understanding of the biological consequences and molecular basis of genetic diseases, particularly those involving errors in large, highly modified molecules such as proteoglycans.

Nanomelic chondrocytes synthesize, but fail to translocate, a truncated aggrecan precursor.

Cartilage extracellular matrix (ECM) is composed primarily of type II collagen and large, link stabilized aggregates of hyaluronic acid and chondroitin sulfate proteoglycan (aggrecan). Maturation and function of these complex macromolecules are dependent upon sequential processing events which occur during their movements through specific subcellular compartments in the constitutive secretory pathway. Failure to complete these events successfully results in assembly of a defective ECM and may produce skeletal abnormalities. Nanomelia is a lethal genetic mutation of chickens characterized by shortened and malformed limbs. Previous biochemical studies have shown that cultured nanomelic chondrocytes synthesize a truncated aggrecan core protein precursor that disappears with time; however, the protein does not appear to be processed by the Golgi or secreted. The present study investigates the intracellular trafficking of the defective aggrecan precursor using immunofluorescence, immunoelectron microscopy and several inhibitors. Results indicate that nanomelic chondrocytes assemble an ECM that contains type II collagen, but lacks aggrecan. Instead, aggrecan precursor was localized intracellularly, within small cytoplasmic structures corresponding to extensions of the endoplasmic reticulum (ER). At no time were precursor molecules observed in the Golgi. In contrast, normal and nanomelic chondrocytes exhibited no difference in the intracellular or extracellular distribution of type II procollagen. Therefore, retention of the aggrecan precursor appears to be selective. Incubation of chondrocytes at 15 degrees C resulted in the retention and accumulation of product in the ER. After a return to 37 degrees C, translocation of the product to the Golgi was observed for normal, but not for nanomelic, chondrocytes, although the precursors disappeared with time. Ammonium chloride, an inhibitor of lysosomal function, had no effect on protein loss, suggesting that the precursor was removed by a non-lysosomal mechanism, possibly by ER-associated degradation. Based on these studies, we suggest that nanomelic chondrocytes are a useful model for examining cellular trafficking and sorting events and the processes by which abnormal products are targeted for retention or degradation. Further investigations should provide insight into the mechanisms underlying chondrodystrophies and other related diseases.

Insulin stimulates mitochondrial protein synthesis and respiration in isolated perfused rat heart.

The rates of synthesis of mitochondrial proteins by both the cytoplasmic and mitochondrial protein synthetic systems, as well as parameters of respiration, were measured and compared in mitochondria isolated from fresh, control perfused, and insulin-perfused rat hearts. The respiratory control ratio (RCR) in mitochondria from fresh hearts was 8.1 +/- 0.4 and decreased to 6.0 +/- 0.2 (P less than 0.001 vs. fresh) in mitochondria from control perfused hearts and to 6.7 +/- 0.2 (P less than 0.005 vs. fresh and P less than 0.02 vs. control perfused) for mitochondria from hearts perfused in the presence of insulin. A positive correlation between the RCR and the rate of mitochondrial translation was demonstrated in mitochondria from fresh hearts. In mitochondria isolated from control perfused hearts, the rate of protein synthesis decreased to 84 +/- 3% of the fresh rate after 30 min of perfusion and fell further to 64 +/- 3% after 3 h of perfusion. The inclusion of insulin in the perfusion buffer stimulated mitochondrial protein synthesis 1.2-fold by 1 h (P less than 0.005) and 1.34-fold by 3 h of perfusion (P less than 0.001). The addition of insulin to 1-h control perfused hearts shifted the rate of mitochondrial protein synthesis from the control level to the insulin-perfused level within 30 min of additional perfusion, whereas 1 h was required to shift the RCR values of these mitochondria from control levels to insulin-perfused levels. Thus, whereas RCR was a useful predictor of mitochondrial translation rates, it did not account for the effects of insulin on mitochondrial translation.(ABSTRACT TRUNCATED AT 250 WORDS)

Coupling of mitochondrial metabolism and protein synthesis in heart mitochondria.

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. In this paper the potential coupling of mitochondrial metabolism to mitochondrial protein synthesis was investigated and the concentration of nucleotides and substrates for optimal translation in isolated rat heart mitochondria was determined. It was demonstrated that optimal isolated heart mitochondrial protein synthesis required the presence of an oxidizable substrate. Of the substrates tested, glutamate (20 mM) supported translation best followed by malate, succinate, and alpha-ketoglutarate, whereas pyruvate supported synthesis poorly. Unlike other recent mammalian mitochondrial systems, the presence of an oxidizable substrate was required for translation even in the presence of medium ATP and an exogenous energy-generating system. Mitochondrial translation also required the presence of adenine nucleotide that could be added as ADP or ATP; however, ATP added above 0.5 mM became progressively inhibitory. As a result, synthesis was supported significantly better by ATP synthesized by the system from added ADP, than by ATP added directly to the system. However, if the phosphorylation of ADP was prevented by limiting the phosphate concentration, ADP itself strongly inhibited mitochondrial protein synthesis. This inhibition appeared to be closely related to the energy charge of the system rather than to absolute levels of ADP, indicating for the first time that mitochondrial translation, like its cytoplasmic counterpart is regulated by energy charge. Last, this system did not require the inhibition of guanine nucleotide or exogenous energy-generating systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and incubation conditions to study heart mitochondrial protein synthesis.

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt. Am. J. Physiol. 258 [Endocrinol. Metab. 21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.