Stephen Keller

Decreased elastic fibers and desmosine content in incompetent cervix

Incompetence of the uterine cervix is a syndrome of painless, progressive dilatation and effacement occurring between the sixteenth and twenty-fourth weeks of gestation that represents abnormal functioning. It may serve as a model to elucidate normal function. Because the incompetent cervix results in painless opening of this organ without uterine contraction before term gestation, it is considered one of the causes of midtrimester spontaneous abortion, habitual spontaneous abortion, and early preterm labor. Untreated, it leads to rapid expulsion and often death of the fetus. We used light microscopy to compare decreased elastic fibers in incompetent cervices with those of normal nonpregnant and pregnant cervices. Morphologic analysis of this difference was extended to biochemical quantification of elastin content in one patient with cervical incompetence. The decrease in elastin suggests that one function of cervical elastin may be to maintain a closed and undilated cervix throughout gestation. There may be a relationship between changes in cross-linked elastin and the incompetent cervix; further studies are therefore indicated.

Solubilized elastin as a substrate for elastase and elastase inhibitor determinations

Abstract This paper describes a test for elastase which employs oxalic acid-solubilized elastin as the substrate. The test described is more sensitive than those using insoluble elastin-dye complexes and more specific than the recently introduced tests using synthetic peptide-esters. It can be used in the presence of other proteins, including serum, and is especially suited for the study of serum elastase inhibitors in health and disease.

Modulation of airspace enlargement in elastase-induced emphysema by intratracheal instillment of hyaluronidase and hyaluronic acid

The study examined how lung hyaluronic acid content influences airspace enlargement in elastase-induced emphysema. To determine the effect of a decrease in hyaluronic acid, hamsters received a single intratracheal instillment of hyaluronidase 24 h prior to administration of pancreatic elastase by the same route. One week later, these animals showed significantly greater airspace enlargement than controls sequentially instilled with saline and elastase (128 vs. 100 microns; p < .05). Conversely, intratracheal administration of hyaluronic acid immediately after elastase instillment resulted in a marked decrease in airspace enlargement at 1 week compared to controls receiving elastase followed by saline (82 vs. 122 microns; p = .005). Since hyaluronic acid has no elastase inhibitory capacity, its effect may involve extracellular matrix interactions not directly related to elastic fiber breakdown. This concept is supported by the finding that animals treated with hyaluronidase and elastase showed no greater loss of lung elastin than that observed in the saline/elastase control group, despite demonstrating a marked increase in airspace enlargement. Further work is needed to determine how hyaluronic acid influences airspace enlargement and to evaluate the potential use of this substance as a treatment for emphysema.

Synthesis of crosslinked elastin by an endothelial cell culture

Summary Synthesis of crosslinked elastin by a major lung cell has not previously been reported. Elastin production by an established clone of rat lung endothelial cells was detected by two separate, highly sensitive methods. The first procedure involved isolation and identification of the labelled, elastin-specific crosslinking amino acids desmosine and isodesmosine by thin layer electrophoresis and radioautography. The second procedure involved detection of elastin by immunofluorescence, using anti-rat lung elastin peptide serum.

The preparation of purified collagenase.

Abstract Two methods are described for the purification of Clostridium histolyticum collagenase. One method involves gel filtration on columns of Sephadex G-200, the other, ion-exchange chromatography on DEAE Sephadex A-50. In both cases, calcium-containing buffers were found necessary for good recoveries of enzyme. Yields of collagenase activity up to 94%, free of nonspecific proteolytic activities, were consistently obtained.

Experimental emphysema in rats: elastolytic titer of inducing enzyme as determinant of the response.

Summary Emphysema-like lesions have been produced in rat lungs by iv administration of various proteolytic enzymes. The severity of the reaction was found to depend on the activity of the enzyme injected. The effect of six microbial proteases was compared in terms of ease of emphysema induction, degree of damage of the fiber structure and accompanying hemorrhage. A positive relationship was established between elastolytic titer of the inducing enzyme and development of emphysematous lesions. On the other hand, hemorrhage was proportional to total proteolytic titer. Best results were obtained when the ratio of specific elastolytic to total proteolytic activity was high. We thank Mr. John Moret for skillful performance of the animal experiments and Miss Mabel Wong for determining enzyme activities.

The content of elastin in the uterine cervix

Mature, crosslinked elastin has been isolated from 4 human and 12 monkey uterine cervices. A modification of previous methods for determination of elastin content was devised to quantitate the low amounts of elastin in the crude connective tissue of uterine cervices. The percentage of elastin was found to range between 0.9 and 2.4% and did not appear to change at various stages of gestation.

Antigenicity and chemical composition of an enzymatic digest of elastin

Abstract Bovine ligamentum nuchae elastin, after solubilization by pancreatic elastase, was dialyzed and fractionated on a column of DEAE-Sephadex A-50. The resulting fractions were analyzed for their component amino acids. The first fraction eluted had the greatest average peptide chain length and 87% of its residues as the three nonpolar amino acids-glycine, valine, and proline in the ratio 2:2:1, with insignificant amounts of the polar amino acids and cross-linking agents. It was found not to have any antigenic sites. Another fraction which was the major component of the digest contained the cross-links in an amount three times that found in complete elastin but was only weakly antigenic. The fraction with the greatest antigenicity was found to be enriched in polar amino acids and tyrosine; it also contained 7% carbohydrate as glucose and 50% lipid.

Microbial Elastases. A Comparative Study.

Summary The effects of pH and of 1% NaCl and human serum on the elastolytic activity of 10 microbial enzyme preparations were determined and compared with those previously reported for pancreatic elastase and Flavobacterium elastase. The pH optima and the NaCl inhibition of the microbial enzymes showed similarity with pancreatic elastase rather than Flavobacterium elastase, but serum inhibition was less pronounced.

Pulmonary Air-Space Enlargement Induced by Intratracheal Instillment of Hyaluronidase and Concomitant Exposure to 60% Oxygen

Although emphysema is generally characterized by damage to pulmonary elastic fibers, the causes of such injury appear to be complex and are not entirely explained by a singular imbalance between elastases and their inhibitors. Other factors could compromise elastic fiber integrity. To test the validity of this argument, hamsters were instilled intratracheally with a nonelastolytic enzyme, hyaluronidase (which reduces lung hexuronic acid content by 21% after 24 h), then exposed to an otherwise nontoxic concentration of oxygen (60%) for 4 days. Additional groups were given (1) hyaluronidase and room air, (2) saline and 60% oxygen, and (3) saline and room air. Treatment with both hyaluronidase and 60% oxygen resulted in a significant increase in air-space enlargement at 4 days (67.1 vs. 57.9 microns for saline/room air controls; p < .05), which was accompanied by only minimal inflammatory changes, as determined by both light microscopy and lavage cytology. Animals receiving either hyaluronidase or 60% oxygen alone showed no significant increases in air-space size compared to those given saline and exposed to room air. While the mechanisms responsible for these results are unclear, the marked increase in radiolabeling of lung elastin cross-links (desmosine and isodesmosine) in animals receiving both hyaluronidase and 60% oxygen (429 vs. 168 cpm/g dry lung for saline/room air controls; p < .05), as well as a significant decrease in total lung desmosine and isodesmosine (32.5 vs. 37.7 micrograms/lung for saline/room air controls; p < .05), suggests that elastic fiber damage is a potential factor. Moreover, only those animals receiving both hyaluronidase and 60% oxygen showed a significant rise in cell-free elastase activity in lavage fluids compared to saline/room air controls (83.3 vs. 48.3 ng; p < .05). On the basis of these findings, it is concluded that while elastic fiber damage may be a common pathway in emphysema, the factors that initiate the disease may be more varied than previously suspected and not always related to the balance between elastases and their inhibitors.