Collagenase from rabbit pulmonary alveolar macrophages.

Rabbit pulmonary alveolar macrophages produce a collagenase which lyses labeled collagen gels, specifically cleaves collagen types I, II and III, is inhibited by ethylenediaminetetraacetate, cysteine, dithiothreitol and serum but is not inhibited by a serine protease inhibitor. Alveolar macrophage collagenase activity can be enhanced by $\text{in vivo}$ BCG activation, $\text{in vitro}$ latex, silica or mycobacterium activation and by $\text{in vitro}$ uncovering of latent enzymatic activity with trypsin treatment. The production of collagenase by unactivated alveolar macrophages and the presence of “latent” collagenase in culture media of alveolar macrophages are examples of significant differences between alveolar and peritoneal macrophages.     

Activation of human breast carcinoma collagenase through plasminogen activator

Latent collagenase activity was detected in the media of a well-characterized line of human breast carcinoma cells maintained for over two years in culture. The media also contained sufficient plasminogen activator to convert extrinsically added plasminogen to plasmin which in turn activated the collagenase. During culture of the breast carcinoma in serum-free medium, collagenase activity was maximum on day 12 whereas plasminogen activator activity changed little with time. Using type I collagen as a substrate, the activated breast tumor collagenase produced $\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments consistent with a mammalian collagenase. These findings suggest a pathologic role of plasminogen activator in the activation of latent collagenase during tumor invasion.A number of investigators have postulated that proteases may play a role in tumor invasion (1–5). Collagenase is one such protease which is active at neutral pH and specifically cleaves triple helical collagen into two ($\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments (6). Secretion of collagenase by tumor cells migrating from the primary mass provides an attractive hypothesis for the mechanism of tumor invasion of surrounding host connective tissue—since the local environment would likely be at neutral pH. Consequently, a number of investigators have reported significant levels of collagenase activity in a wide variety of tumors (7–14). Abramson (13) has correlated aggressive $\text{in vivo}$ growth in carcinomas of the head and neck with collagenase activity, and Kuettner et al. (14) have postulated that inhibitors of collagenase may prevent tumors from invading cartilage.Collagenase is produced in both latent and active forms (6). The latent form can be activated with brief protease treatment (15). Since one of the proteases capable of activating collagenase is plasmin (15), the possibility arose that tumor cells could activate collagenase through plasminogen activator. Plasminogen activator secreted by tumor cells (4, 5) could convert plasminogen zymogen to plasmin which would in turn activate latent tumor collagenase. Testing this hypothesis $\text{in vitro}$ was the subject of the present study.Previous studies on collagenase from human carcinoma (7, 13, 14) have suffered from the drawback that contaminating inflammatory cells and fibroblasts may have been the source of the collagenase. Therefore, we have studied collagenase production from cultured human breast carcinoma cells which have been well characterized to be mammary epithelial in origin, malignant in karyotype, and able to grow in nude mice. Production of collagenase from these cells is therefore unequivocally of human carcinoma origin. The time course of latent collagenase and plasminogen activator secretion by these cultured tumor cells was studied following withdrawal of serum. To test whether plasminogen activator was secreted in sufficient amounts to indirectly activate latent collagenase, collagenase activity of the culture media was studied after the extrinsic addition of plasminogen. Finally, to verify that the tumor-secreted collagenase cleaved type I collagen at a single locus, enzyme degradation products were studied by gel electrophoresis.     

Reversible inactivation of soluble liver guanylate cyclase by disulfides

A new amino acid was isolated from the cuticle collagen of $\text{Ascaris lumbricoides}$ and characterized by ultraviolet, mass and nmr spectroscopies and chemical degradation. The results indicate that the compound is an isomer of trityrosine, having an ether linkage. The name “isotrityrosine” is proposed. Its structure suggests that it serves as a crosslink and plays a role in the organization of the collagen structure.     

Carbohydrate structure and serological behaviour of "antifreeze" glycoproteins from an Antarctic fish.

The carbohydrate moiety of the “antifreeze” glycoprotein from $\text{Trematomus borchgrevinki}$ was found to be β-D-galactosyl 1–3 N-acetyl galactosamine by gasliquid chromatography. The glycoprotein inhibited anti-T antibody from human serum and $\text{Arachis hypogoea}$ lectin, but was inactive against $\text{Vicia graminea}$. Native “antifreeze” glycoprotein did not inhibit the agglutinins from $\text{Helix pomatia}$ or $\text{Cepaea hortensis}$, although after Smith degradation showed a strong inhibition towards them. Inhibition of the latter agglutinin demonstrates the carbohydrate-protein linkage to be α-linked. The presence of the Thomsen-Friedenreich antigen (T-antigen) on the “antifreeze” glycoprotein and its relation to tumour cell surfaces is briefly discussed.     

Stereochemistry of leukotriene C-1

Leukotriene C-1, a “Slow Reacting Substance” (SRS), has been shown to possess the molecular Structure depicted by V (5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid) by its identity with a totally synthetic product of known structure and stereochemistry.     

Cellular compartmentation of two species of δ-aminolevulinic acid synthetase in a facultative photohetero-trophic bacterium (Rps. spheroides Y.)

Both species of δ-aminolevulinic acid (ALA) synthetase appear compartmentalized in $\text{Rps. spheroides}$ Y. The ALA synthetase, “F_I”, activity is correlated with dark respiratory metabolism and is a cytoplasmic “soluble” enzyme. The other enzyme, “F_II”, induced only in light, is in chromatophores and its activity is correlated with photometabolism.     

Cloning of the replication origin from Drosophila virilis mitochondrial DNA.

Mitochondrial DNA from $\text{Drosophila}$ contains high “A+T”-rich region. Its DNA replication starts in the “A+T”-rich region and proceeds unidirectionally around the molecule. In order to determine precise location of the DNA replication origin and elucidate unique feature of its nucleotide sequence, the “A+T”-rich region of mitochondrial DNA from $\text{Drosophila}\text{virilis}$ has been cloned in $\text{Escherichia}\text{coli}$. The chimeric plasmid DNA containing the “A+T”-rich region stimulates $\text{in}\text{vitro}$ DNA replication system from $\text{Drosophila}\text{virilis}$ mitochondria about ten fold higher than the parental plasmid DNA, as does native mitochondrial DNA.     

Sequential homology of the collagenase from eucaryote Hypoderma lineatum with the proteinases of the trypsin family

The sequence of 32 N-terminal amino acid residues of the collagenase from the larvae Hypoderma lineatum of mol. weight 24000 is homologous with the sequences of pancreatic proteinases. This analysis confirms the hypothesis of similarity made on the basis of amino acid composition, physico-chemical properties and inhibition studies. It is proposed that within the trypsin-like family exists a group of eucaryote collagenases with digestive rather than morphogenic function which cleave specifically native collagen at $\text{3}\text{4}$ from its N-terminus.     

Excision of thymine dimers by proteolytic and amber fragments of E. coli DNA polymerase I

Excision of thymine dimers from specifically incised ultraviolet irradiated DNA by $\text{E. coli}$ DNA polymerase I is stimulated by concurrent DNA synthesis. The 36,000 molecular-weight “small fragment” obtained by limited proteolysis of DNA polymerase I, which retains only the 5′ → 3′ exonuclease activity, also excises thymine dimers, but at one-tenth the rate of the intact enzyme. However, the rate of excision is increased by addition of the “large” 76,000-molecular weight fragment. With the further addition of the 4 deoxynucleoside triphosphates, permitting DNA synthesis to occur, excision approaches rates observed with the intact enzyme. The same result was obtained with a fragment of DNA polymerase I with 5′ → 3′ exonuclease activity that is present uniquely in polymerase I $\text{amber}$ mutants.     

Hormonal regulation of macrophage collagenase activity.

Whereas peritoneal macrophages from nonpregnant guinea pigs were stimulated $\text{in vitro}$ by endotoxin to produce collagenase on the second day of culture, those from pregnant guinea pigs were incapable of this response. However, if the cells from pregnant animals were preincubated for one day prior to endotoxin stimulation, collagenase activity could be detected. Injection of either estrogen or progesterone into guinea pigs at doses comparable to those found during pregnancy prior to removal of the peritoneal cells also inhibited the $\text{in vitro}$ stimulation of collagenase production. The addition of these hormones $\text{in vitro}$ revealed that at 5 × 10^?6 M estrogen and progesterone inhibited 53% and 100% respectively of the collagenase activity. Addition of both hormones at a final concentration of 5 × 10^?7 M of each inhibited 87% of the activity indicating a synergistic effect since this concentration of either hormone alone was ineffective.     

Dental pulp collagenase: initial demonstration and characterization.

A collagenase, active against native helical collagen, was initially found in the explant medium of bovine dental pulp. In contrast to the collagenases from other oral tissues, all the pulp enzyme released was in a latent form which was activated by trypsin treatment, 4-aminophenylmercuric acetate, and some chaotropic agents. The activated enzyme was inhibited by low concentrations of EDTA and calf serum. The molecular weight of activated enzyme was tentatively estimated at 45,000 daltons by gel filtration. The enzyme attacked undenatured collagen in solution at 20°C producing characteristic products $\text{α}{}^{\mathrm{A}}\text{(}\text{3}\text{4}\text{)}$ and $\text{α}{}^{\mathrm{B}}\text{(}\text{1}\text{4}\text{)}$.     

A difference in the affinity labeling of Ca2+, Mg2+-activated ATPases of normal and unc A strains of Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate

The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca²⁺, Mg²⁺-activated ATPase of $\text{Escherichia}\text{coli}$. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an $\text{unc A}$ mutant of $\text{E.}\text{coli}$ was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.     

Indole alkaloid biosynthesis: Partial purification of “ajmalicine synthetase” from Catharanthus roseus

The “ajmalicine synthetase” system of $\text{Catharanthus roseus}$ has been partially purified from callus, seedlings and mature plants. On gel filtration of the cell-free extract, four β-D-glucosidase isozymes were observed in seedlings and plants. Only two were present in the callus. A protein peak at 55,000 daltons in all three materials was capable of synthesizing ajmalicine from tryptamine and secologanin in the presence of NADPH. This “ajmalicine synthetase” rapidly lost its ability to synthsize ajmalicine, but retained the β-glucosidase activity.     

An in vitro model for the study of collagen degradation during acute inflammation

Collagen sponges, labelled with fluorescein, were implanted under the back skin of sensitized rats. These elicited an acute inflammatory reaction with cellular invasion of the sponge and the development of a fibrous capsule at the periphery. After 4 days each sponge, together with the fibrous capsule, was excised and placed into tissue culture. Degradation of the collagen sponge by the invading cells was monitored from the release of soluble fluorescein peptides into the medium. The addition of foetal calf serum caused inhibition above 5% (v/v). Inhibitors of collagenase and neutral proteinases blocked the release of fluorescein peptides. Collagenolysis was also abolished or retarded by inhibitors of lysosomal cathepsins. The anti-inflammatory drug, dexamethasone, blocked all collagenolytic activity whereas indomethacin was without effect. The $\text{ex}$$\text{vivo}$ model offers the possibility for following the activity of the invading phagocytic cells and for examining the enzymatic mechanisms involved in collagenolysis using appropriate perturbation techniques.     

Effect of vitamin B-12 deprivation on the rates of synthesis and degradation of rat liver fatty acid synthetase

To characterize the basis for the increased hepatic fatty acid synthetase activity in vitamin B-12 deprivation, the content and rates of synthesis and degradation for the enzyme were determined. Animals were in a dietary steady state on normal chow or a B-12-deprived diet; animals on the latter diet were further divided into a “supplemented” group given B-12 and those “B-12-deprived.” The B-12-deprived animals had tissue B-12 depletion. Both total and specific activity of fatty acid synthetase were increased in the B-12-deprived animals, and this was due to increased enzyme protein. Rates of synthesis and degradation were studied in each group after a pulse of 20 μCi of l-[U-¹⁴C]leucine. Radioactivity was determined in the immunoprecipitate of the purified enzyme. Relative rates of synthesis in the B-12-deprived group were increased 8.8-fold over the normal and 3.6-fold over the B-12-supplemented group. Degradation of hepatic fatty acid synthetase was 63 hr ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) in the normal and 65 hr in the B-12-supplemented group. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ in the B-12-deprived group was 35 hr. Degradation rates for the soluble protein pool were not affected by B-12 deprivation. The rate constant for synthesis of hepatic fatty acid synthetase in the B-12-deprived group was 14-fold that of the normal and 6-fold that of the B-12-supplemented animals. Thus, although vitamin B-12 deprivation results in increased rate of degradation of fatty acid synthetase, enzyme synthesis is markedly increased yielding a severalfold net increase in synthetase content and activity.     

Separation of acetanilide and its hydroxylated metabolites and quantitative determination of "acetanilide 4-hydroxylase activity" by high-pressure liquid chromatography.

A simple and very sensitive method for the separation of 4-hydroxyacetanilide, 3-hydroxyacetanilide, 2-hydroxyacetanilide, and acetanilide was developed with the use of high-pressure liquid chromagraphy. Each of these phenolic derivatives can be separated completely from acetanilide and from one another. A simple assay for “acetanilide 4-hydroxylase activity” is thus described. The limit of sensitivity for cytochrome P-450-mediated acetanilide 4-hydroxylase activity is estimated to be 1.0 pmol/min/mg microsomal protein, thereby allowing this assay to be useful in detecting monooxygenase activity in “low level” nonhepatic tissues. Hepatic acetanilide 4-hydroxylase activity is induced about fourfold in $\text{C57BL}\text{6N}$ mice by 3-methylcholanthrene. Although acetanilide 2-hydroxylase activity is about seven times lower than the 4-hydroxylase activity, the 2-hydroxylase is also induced about three- or fourfold in $\text{C57BL}\text{6N}$ mice by 3-methylcholanthrene. The “2-hydroxylase activity” cannot, however, be strictly quantitated under the conditions described herein. The K_m values of both the 3-methylcholanthrene-induced and control 4-hydroxylase activity are about 0.55 mm; V_max values for 3-methylcholanthrene-treated and control mice, respectively, are 4.9 ± 1.1 and 1.1 ± 0.31 nmol/min/mg microsomal protein. The 4-hydroxylase in the liver of both 3-methylcholanthrene-treated and control mice appears to represent two or more catalytic activities, i.e., two or more forms of P-450 having widely differing affinities for the substrate acetanilide.     

Reiteration of DNA complementary to a cytoplasmic non-ribosomal RNA

Experimentally induced granulomas, in guinea pigs, were fractionated into a 40,000xg pellet and supernatant, which was further fractionated into a 100,000xg pellet and supernatant. The product of in vitro amino acid incorporation by the 40,000xg pellet was tentatively identified as collagen by its high proline/leucine ratio, its digestibility by bacterial collagenase and its solubility in hot trichloroacetic acid. The 100,000xg pellet incorporated leucine much more efficiently than the 40,000xg pellet and the product was insoluble in hot trichloroacetic acid.Labeled RNA from the 40,000xg pellet formed hybrids with granuloma ($\text{C}{}_{\mathrm{o}}\text{t}\text{1}\text{2}\text{= 100–150}$) and liver ($\text{C}{}_{\mathrm{o}}\text{t}\text{1}\text{2}\text{= 8,000}$) $\text{chromatin}$ DNA, indicating that genes coding for this RNA are repeated about 100-fold in granuloma and less than 5 times in liver DNA. Under conditions of poly(A) binding, 50% of this labeled RNA is retained by filters. Digestion with ribonuclease and ribonuclease T₁, decreases binding efficiency by 75%.     

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-¹⁴C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the $\text{in situ}$ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in $\text{vitro}$ by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.     

Ultraviolet light-induced recombination

Stimulation of transduction in $\text{Escherichia coli}$ by ultraviolet irradiation of the transducing phage P1 requires the $\text{uvrA-uvrB}$ nuclease but not the $\text{uvrC}$ product or DNA polymerase I. It is hypothesized that the first step in “normal” recombination can be bypassed by any procedure generating single-stranded ends of DNA (as, for example, by $\text{uvra-uvrB}$ nuclease activity).     

Control of pattern duplication in the retinotectal system of Xenopus. Induction of duplication in eye fragments by secondary cuts.

Following surgical ablation of the temporal (posterior) region of the eye-bud in stage 32 Xenopus frog embryos, the surviving nasal (anterior) fragment gradually rounds up to form a functional eye and orderly retinotectal map. Large nasal fragments ($\text{N}\text{-}\text{2}\text{3}$) assemble topographically normal maps, as does the majority of nasal “half-eye” fragments; small nasal fragments ($\text{N}\text{-}\text{1}\text{3}$), and a minority of nasal half-eye fragments, give a characteristic, mirror-symmetrical duplication map, similar (but not identical) to the “double-nasal” maps which develop when two nasal half-eyes are fused to form a frank NN double-eye. Ventral fragments and temporal fragments show similar size-dependent behavior, although their characteristic duplicate maps are topographically different from those of nasal fragments and more similar to the “double-ventral” and “double-temporal” maps of VV and TT recombinant eyes. Here we show that a simple surgical transection, applied either dorsally or ventrally to large nasal ($\text{N}\text{-}\text{2}\text{3}$) fragments so as to isolate a subregion of the tissue at the dorsum or venturm of the fragment, induces full or partial duplication of the nasal type in the majority of cases. The results refute the hypothesis that special properties at the eye-bud center, by their presence or absence in the fragment, control pattern duplication, and point instead toward interactions around the circumference of the eye-bud as a crucial parameter in determining positional information in the retina.