Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.     

Protocol for sampling environmental sites for legionellae.

A protocol for sampling environmental sites was developed and used to identify possible sources of Legionella species in support of epidemiologic investigations at two hospitals. In hospital A, legionellae were isolated from 43 of 106 (40%) different sites. Three separate Legionella pneumophila serotypes and a previously unrecognized species were present in different combinations in the positive samples. Two of five cooling towers contained the same L. pneumophila serogroup 1 monoclonal type (1,2,4,5) as was isolated from patients. The same monoclonal type was also isolated from make-up water for the two cooling towers, a hot water tank, water separators in four main air compressor systems for respiratory therapy, and cold and hot water faucets. In hospital B, 13 of 37 (38%) sample sites contained legionellae, all of which were L. pneumophila serogroup 1. The monoclonal type matching isolates from patients (1,2,4,5) was found at the highest concentration in a hot water tank, but it was also present at four other sample sites. Since legionellae not related to disease may be found in many of the sites sampled, an epidemiologic association with the probable source should be established before intervention methods, such as disinfection, are undertaken.     

Active site of human liver aldehyde dehydrogenase

Bromoacetophenone (2-bromo-1-phenylethanone) functions as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) and has been found specifically to label a unique tryptic peptide in the enzyme. Amino-terminal sequence analysis of the labeled peptide after purification by two different procedures revealed the following sequence: Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys. Radioactivity was found to be associated with the glutamate residue, which was identified as Glu-268 by reference to the known amino acid sequence. This paper constitutes the first identification of an active site of aldehyde dehydrogenase.     

Functional differences between small and large luteal cells of the late-pregnant vs. nonpregnant cow

This paper describes an in vitro model for the study of two types of steroidogenic luteal cells from cows in different physiological states. Two different populations of enzymatically dispersed bovine luteal cells were separated on the basis of size in a Cel-Sep Sedimentation Chamber. The separated small (12.5-23 micron in diameter) and large (greater than 23 micron in diameter) luteal cells of late-pregnant cows (Days 190-280) contained the distinct morphological characteristics previously defined for these two populations of cells. Cells were evaluated for progesterone (P4) production during a 3-h incubation with and without bovine luteinizing hormone (bLH, 10 ng/ml). Both small and large luteal cells from the late-pregnant cow were found to contain equal levels of P4 at Time 0 and increased but equal levels of P4 after a 3-h incubation. Neither cell type showed an increase in P4 production in response to the addition of bLH (p greater than 0.05). Since these results differed from earlier reports for luteal cells of the nonpregnant cow, small and large luteal cells of the mid-cycle (Day 14) were incubated, and the levels of P4 production were compared with P4 levels from the late pregnant cow. In agreement with previous reports for nonpregnant cows, progesterone content at Time 0 was 7-fold higher in large cells than in small cells (p less than 0.05), and after 3 h of incubation, 13-fold higher (p less than 0.05). Although the small cells responded to the presence of bLH in the incubation medium with a 4-fold increase in P4 production, this increase was not significant (p greater than 0.05). The large cell did not respond to bLH. However, the large cell type continued to contain and produce more P4 than did the small cells treated with bLH. This study indicates that both the small and large luteal cells of late-pregnancy are able to produce P4. However, the large luteal cell of the estrous cycle produces greater quantities of P4 than does the small luteal cell or the large luteal cell of late pregnancy.     

Techniques in molecular biology

Book Review

Techniques in molecular biologyJ.M. Walker and W. Gaastra (Eds.), vol. 2. London: Croom Helm, 1987. iv + 332 pages. £14.95. ISBN 0-7099-3673-7     

The effect of vitamin E on lipid peroxidation in the copper-deficient rat

The present study was designed to determine whether the supplementation of vitamin E in the copper-deficient diet would ameliorate the severity of copper deficiency in fructose-fed rats. Lipid peroxidation was measured in the livers and hearts of rats fed a copper-deficient diet (0.6 microg Cu/g) containing 62% fructose with adequate vitamin E (0.1 g/kg diet) or supplemented with vitamin E (1.0 g/kg diet). Hepatic lipid peroxidation was significantly reduced by vitamin E supplementation compared with the unsupplemented adequate rats. In contrast, myocardial lipid peroxidation was unaffected by the level of vitamin E. Regardless of vitamin E supplementation, all copper-deficient rats exhibited severe signs of copper deficiency, and some of the vitamin E-supplemented rats died of this deficiency. These findings suggest that although vitamin E provided protection against peroxidation in the liver, it did not protect the animals against the severity of copper deficiency induced by fructose consumption.     

A major 62-kD intranuclear matrix polypeptide is a component of metaphase chromosomes

We have isolated and partially characterized a major intranuclear matrix polypeptide from rat liver. This polypeptide, which is reversibly stabilized into the intranuclear matrix under conditions which promote intermolecular disulfide bond formation, has a Mr of 62,000 and pI of 6.8-7.2 as determined by two-dimensional IEF/SDS-PAGE. A chicken polyclonal antiserum was raised against the polypeptide purified from two-dimensional polyacrylamide gels. Affinity-purified anti-62-kD IgG was prepared and used to immunolocalize this polypeptide in rat liver tissue hepatocytes. In interphase hepatocytes the 62-kD antigen is localized in small, discrete patches within the nucleus consistent with the distribution of chromatin. The staining is most prominent at the nuclear periphery and somewhat less dense in the nuclear interior. Nucleoli and cytoplasm are devoid of staining. During mitosis the 62-kD antigen localizes to the condensed chromosomes with no apparent staining of cytoplasmic areas. The chromosomal staining during mitosis is uniform with no suggestion of the patching seen in interphase nuclei. Fractionation and immunoblotting studies using rat hepatoma tissue culture cells blocked in metaphase with colcemid confirm the chromosomal localization of this 62-kD intranuclear protein during mitosis. The 62-kD polypeptide fractionates completely with metaphase chromosome scaffolds generated by sequential treatment of isolated chromosomes with DNAse I and 1.6 M NaCl, suggesting that this major 62-kD intranuclear protein may be involved in maintaining metaphase chromosomal architecture.     

Characterization of the DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin

The DNA-binding properties of the receptor for 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G-25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or alpha-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre-treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions.     

Syngeneic monoclonal internal image anti-idiotopes as prophylactic vaccines

A syngeneic monoclonal anti-idiotope that behaves as an internal image of the mammalian reovirus type 3 cellular attachment protein (viral hemagglutinin) was used in the syngeneic host for the induction of a prophylactic anti-viral antibody response. These studies were performed without the aid of co-stimulation by viral antigens. The high stringency of this system enables us to define the maximum constraints on the use of anti-idiotopes as anti-viral vaccines. We have used the murine BALB/c monoclonal IgM anti-idiotope 87.92.6 to study the idiotope and antigen specificity, kinetics, dose dependence, adjuvant, carrier, and valency requirements of anti-idiotope-induced anti-viral antibody responses. These studies show that the production of high titer neutralizing antibody requires a lengthy (60 day) immunization protocol, which includes the use of adjuvant and multivalent anti-idiotope, and is dependent on anti-idiotope concentrations of greater than 50 micrograms. When administered in this manner anti-idiotope can stimulate serotype-specific antibody responses across species barriers at levels comparable with those obtained after inoculation with virus. The practical efficacy of these reagents and procedures is documented by the ability of maternal immunization with anti-idiotope to confer complete protection in neonates from a potentially lethal reovirus type 3 viral infection.