Altered posttranslational modifications of collagen in keloid.

Keloid is a tissue with an excessive accumulation of collagen. In this study, we have partially characterized post-translational modifications of type I collagen in human keloid in order to pursue their potential involvement in this pathology. The levels of lysyl hydroxylation of the helical portions of alpha 1 and alpha 2 chains of type I collagen in keloid were significantly higher than those of normal, while the levels of prolyl hydroxylation were identical between these two groups. The contents of the major reducible cross-links in dermal collagen, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymero-desmosine, were both significantly higher in keloids (up to sixfold) than those of normal. In addition, significant amounts of hydroxylysine-aldehyde derived cross-links that are characteristic of skeletal tissue collagens, dehydro-dihydroxylysinonorleucine (about 0.3 mole/mole of collagen) and pyridinoline (about 0.1 mole/mole of collagen), were found in keloids. These results indicate that keloid-forming cells are phenotypically different from those in normal dermis and that the collagen produced is highly cross-linked. The increased cross-linking provides the fibrils with more stability that may result in an accumulation of collagen.

Mainstream end-tidal carbon dioxide monitoring in the neonatal intensive care unit.

BACKGROUND: Continuous noninvasive monitoring of arterial carbon dioxide (CO2) in neonatal intensive care unit (NICU) patients would help clinicians avoid complications of hypocarbia and hypercarbia. End-tidal CO2 monitoring has not been used in this population to date, but recent technical advances and the introduction of surfactant therapy, which improves ventilation-perfusion matching, might improve the clinical utility of end-tidal monitoring. OBJECTIVE: To determine the accuracy and precision of end-tidal CO2 monitoring in NICU patients. DESIGN: Nonrandomized recording of simultaneous end-tidal and arterial CO2 pairs. SETTING: Two university NICUs. PATIENTS: Forty-five newborn infants receiving mechanical ventilation who had indwelling arterial access, and a predefined subsample of infants who were <1000 g birth weight, <8 days of age, and who received surfactant therapy (extremely low birth weight -ELBW- <8). OUTCOME MEASURES: The correlation coefficient, degree of bias, and 95% confidence interval were determined for both the overall population and the ELBW <8 subgroup. Those factors which significantly influenced the bias were identified. The ability of the end-tidal monitor to alert the clinician to instances of hypocarbia or hypercarbia was determined. RESULTS: There were 411 end-tidal/arterial pairs analyzed from 45 patients. The correlation coefficient was 0.833 and the bias was -6. 9 mm Hg (95% confidence interval, +/-11.5 mm Hg). The results did not differ markedly in the ELBW <8 infants. Measures of the degree of lung disease, the ventilation index and the oxygenation index, had small influences on the degree of bias. This type of capnometry identified 91% of the instances when the arterial CO2 pressure was between 34 and 54 mm Hg using an end-tidal range of 29 to 45 mm Hg. End-tidal values outside this range had a 63% accuracy in predicting hypocarbia or hypercarbia. CONCLUSION: End-tidal CO2 monitoring in NICU patients is as accurate as capillary or transcutaneous monitoring but less precise than the latter. It may be useful for trending or for screening patients for abnormal arterial CO2 values.

The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system.

This study reports the expression of functional human lysyl hydroxylase (LH), a post-translational modifying enzyme that catalyzes the hydroxylation of the lysine residues essential for cross-linking in collagen biosynthesis. We have developed a novel baculovirus system for the expression of LH, a protein that exists normally within the lumen of the endoplasmic reticulum, using a powerful baculovirus signal sequence for secretion. The supernatant from Sf9 cells infected with the viral recombinant showed significant LH activity that increased linearly with supernatant concentration, whereas there was no detectable LH activity in the cell pellet. Silver staining of the fractions purified from the active supernatant by concanavalin A Sepharose chromatography and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated an 85-kDa protein (the expected size of the LH subunit) that was most prominent in those fractions with the highest LH activity. N-terminal amino acid sequencing verified that the N-terminal primary structure of this 85-kDa protein was identical to human LH. Moreover, the activity of the expressed protein was shown to be dependent on the presence of Fe++, ascorbate, and alpha-ketoglutarate, three essential cofactors for LH activity. We have therefore successfully developed a novel expression system that produces functional human LH and enables this normally nonsecretory enzyme to be secreted, facilitating its separation from the intracellular proteins of insect cells. Future applications should allow characterization of the LH active site by crystallographic studies and site-directed mutagenesis for structure-function comparison.

The mRNA and the activity of lysyl hydroxylase are up-regulated by the administration of ascorbate and hydralazine to human skin fibroblasts from a patient with Ehlers-Danlos syndrome type VI.

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine required for the intermolecular cross-linking of collagen, which is an essential step in collagen biosynthesis. Dermal fibroblasts from patients with Ehlers-Danlos Syndrome type VI (EDS VI), an inherited connective tissue disorder, express decreased levels of LH activity. In the present study we have shown that both the mRNA and the enzyme activity of LH in skin fibroblasts from one EDS VI patient (AT750), a compound heterozygote for the LH gene, are increased by administration of ascorbate and hydralazine, either individually or in combination. Although the AT750 cells express only 24% of the LH activity found in normal cells, a similar fold increase in activities in both EDS VI and normal cells was observed following treatment with ascorbate (1.5- to 2-fold) and hydralazine (2- to 4-fold), which paralleled the increase in their steady state LH mRNAs. Ascorbate increased total collagen production by 2-fold from a similar basal level of collagen synthesis in each cell type. This was confirmed by protein gel analysis which showed increases in pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen chains in both normal and EDS VI cells. This ascorbate-mediated increase of alpha 1(I) collagen resulted from increased mRNAs for alpha 1(I) collagen in both cell types. Hydralazine treatment, with or without ascorbate, severely decreased the alpha 1(I) collagen mRNAs in fibroblasts from both AT750 and the normal donor; total collagen synthesis was similarly reduced. This study shows that LH activity, which is severely deficient in fibroblasts from an EDS VI patient, can be upregulated by administration of ascorbate and hydralazine as a result of the increased mRNA for LH, suggesting that the mechanism for the regulation of the LH gene is functioning normally in this patient.

Leukemia inhibitory factor induces changes in lipid metabolism in cultured adipocytes.

Cytokines induce a number of changes in lipid metabolism that can produce hyperlipidemia. Leukemia inhibitory factor (LIF), a recently discovered cytokine, has been suggested to play a role in the cancer cachexia syndrome through its ability to decrease lipoprotein lipase (LPL) activity. This study explores the mechanism by which LIP decreases LPL activity in cultured adipocytes and determines its effects on fatty acid synthesis and lipolysis to see if it shares the same catabolic effects on fat cells as seen with other cytokines, such as tumor necrosis factor (TNF). LIF decreased LPL activity in cultured adipocytes by 44% compared with an 85% decrease produced by TNF. Although the percent decrease in LPL activity is not as great in LIF-incubated adipocytes as in TNF-incubated adipocytes, the half-maximal doses for both cytokines are similar. LPL messenger RNA levels paralleled LPL activity in the LIF-treated adipocytes, suggesting that the effect of LIF on LPL activity is predominantly mediated through transcriptional regulation. In contrast to TNF, LIF tended to increase the de novo synthesis of fatty acids. Acetyl coenzyme-A carboxylase messenger RNA levels paralleled the changes seen in fatty acid synthesis for both cytokines. LIF caused a small increase in lipolysis, whereas TNF increased lipolysis by greater than 2-fold. These results demonstrate that the catabolic effects of LIF are weaker than those of TNF and are predominantly directed toward decreasing LPL activity, which may contribute to the hyperlipidemia associated with infection, inflammation, and cancer.

A patient with Ehlers-Danlos syndrome type VI is a compound heterozygote for mutations in the lysyl hydroxylase gene.

In the present study, we have isolated and sequenced the complementary DNAs of two mutant alleles for lysyl hydroxylase (LH) in fibroblasts from one patient (AT750) with Ehlers-Danlos syndrome type VI (EDS VI). We have identified a putative mutation in each allele which may be responsible for the patient's decreased LH (normalized to prolyl hydroxylase) activity (24% of normal). Intermediate levels of LH activity were measured in the patient's parents, who are clinically normal (father 52%; mother 86%). After the cloning of cDNAs and amplification by PCR, sequence analysis revealed two equally distributed populations of cDNAs for LH in the AT750 cell line. Each allele revealed different but significant changes from the normal sequence. In one allele (allele 1), the most striking change was a triple base deletion that would result in the loss of residue Glu532. The most significant difference in the other allele (allele 2) was a G-->A change which would produce a Gly678-->Arg codon change in a highly conserved region of the enzyme. Restriction analysis identified that allele 1 was inherited from the proband's mother and allele 2 from the father. This study represents the first example of compound heterozygosity for the LH gene in an EDS VI patient, and it appears that there is an additive effect of each mutant allele on clinical expression in this patient.

Sequence analysis of a cDNA for lysyl hydroxylase isolated from human skin fibroblasts from a normal donor: differences from human placental lysyl hydroxylase cDNA.

Using polymerase chain reaction, we have isolated and sequenced a 3-kb cDNA for lysyl hydroxylase (LH) from human skin fibroblasts from an normal donor. Apart from two polymorphic sites, no differences were observed between the 2184 nt coding regions of LH cDNA from fibroblasts and placenta. However, four differences were observed in the 3' non-coding regions of the two cDNAs; three were single base changes and the fourth a deletion of a single base. The absence of the single nucleotide in the LH cDNA from fibroblasts resulted in the loss of an HpaII site that is present in the placental LH cDNA; this was confirmed in HpaII digests of fibroblast and placental LH cDNAs from the same donor. Northern blots showed that the LH gene was strongly expressed in fibroblasts and placenta and, to a lesser extent, in aorta, lung, vein, cartilage, and artery.

Regulation of lysyl oxidase mRNA in dermal fibroblasts from normal donors and patients with inherited connective tissue disorders.

Lysyl oxidase (LO) is an extracellular copper-dependent enzyme that catalyzes the initial reaction in the formation of lysine or hydroxylysine-derived crosslinks during collagen biosynthesis. We have isolated a cDNA for human LO from skin fibroblast poly(A+)RNA by PCR using primers based on the recently published sequence of human LO. This cDNA probe detects a major mRNA of 4.2 kb on Northern blots of RNA from normal fibroblasts. The level of LO mRNA was not significantly affected by cell density or by ascorbate treatment. Treatment of skin fibroblasts with hydralazine (50 microM), which increases the mRNAs for both the alpha and the beta subunits of prolyl hydroxylase (PH) and the mRNAs for lysyl hydroxylase, also increased LO mRNA by fourfold over a 72-h time course. In contrast, hydralazine dramatically decreased the mRNAs for alpha 1(I) collagen. Administration of minoxidil (500 microM), which specifically decreases LH activity without affecting PH activity or collagen biosynthesis in skin fibroblasts, stimulated the level of LO mRNA. Neither the administration of penicillamine (100 microM), which interferes with collagen cross-linking, nor the administration of beta-aminopropionitrile, which is a strong irreversible inhibitor of LO, to fibroblasts significantly changed the levels of LO mRNA over a 72-h time course. However, bleomycin (0.6 microgram/ml) significantly decreased the 4.2-kb LO mRNA in contrast to the levels of the alpha 1(I) collagen mRNAs, which were unchanged. No significant change was observed in the steady-state levels of LO mRNAs in fibroblasts isolated from patients with certain connective tissue disorders, including Marfan syndrome, Menkes disease, cutis laxa, and pseudoxanthoma elasticum.

Acute adrenal insufficiency in association with pancreatic carcinoma.

Addison's disease secondary to metastatic cancer to the adrenal gland is underdiagnosed. Prompt diagnosis and treatment is essential and could enhance the quality of life. Cases of adrenal insufficiency produced by metastatic carcinoma are unusual, despite the frequency of carcinomatous metastases to the adrenal glands. The clinical features of adrenal insufficiency are relatively nonspecific and can be easily overlooked in a patient with a malignant neoplasm. We report herein the case of a middle-aged man who presented with adrenal insufficiency in association with pancreatic carcinoma. To our knowledge, this is the first reported case of adrenal insufficiency occurring with adenocarcinoma of the pancreas.

Comparison of once-daily captopril or enalapril in mild essential hypertension.

The purpose of this study was to assess the effect of a daily low dose of the angiotensin-converting enzyme (ACE) inhibitors, captopril or enalapril, in mild essential hypertension. Nine men with seated diastolic blood pressure between 95 and 104 mm Hg on placebo participated in the study. After one month of placebo, captopril 25 mg was administered; blood pressure, heart rate, ACE activity and plasma renin activity were measured hourly for 4 hours. Each patient then received captopril 50 mg once daily for 8 weeks and similar measurements were made 24 hours post-dose every 2 weeks. After another month of placebo, the identical protocol was repeated after enalapril 5 mg. Although blood pressure and ACE activity decreased significantly (P less than 0.05) within 2-4 hours of the acute doses of each inhibitor, neither captopril or enalapril produced significant reductions 24 hours after the small daily dose. Thus, neither ACE inhibitor alone was adequate to control blood pressure in mild hypertension when given once daily during 8 weeks of treatment.