Processing of Laminin-5 and Its Functional Consequences: Role of Plasmin and Tissue-type Plasminogen Activator

The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell– matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5–rich matrices of pp126 epithelial cells fail to assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126 cells, using a panel of laminin-5 subunit-specific antibodies. The α3 subunit of laminin-5 secreted by pp126 cells migrates at 190 kD, whereas that secreted by 804G and MCF-10A cells migrates at 160 kD. The pp126 cell 190-kD α3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5 β and γ chains. After plasmin treatment, pp126 cell laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported by immunofluorescence analyses that demonstrate colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells. Whereas tissue-type plasminogen activator (tPA), which converts plasminogen to plasmin, codistributes with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5–rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5 α3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as an adhesive factor. In cells in culture, this functional conversion appears dependent upon and is regulated by tPA and plasminogen.     

Herbicide Safener-Binding Protein of Maize : Purification, Cloning, and Expression of an Encoding cDNA

Dichloroacetamide safeners protect maize (Zea mays L.) against injury from chloroacetanilide and thiocarbamate herbicides. Etiolated maize seedlings have a high-affinity cytosolic-binding site for the safener [³H](R,S)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazol-idine ([³H]Saf), and this safener-binding activity (SafBA) is competitively inhibited by the herbicides. The safener-binding protein (SafBP), purified to homogeneity, has a relative molecular weight of 39,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 5.5. Antiserum raised against purified SafBP specifically recognizes a 39-kD protein in etiolated maize and sorghum (Sorghum bicolor L.), which have SafBA, but not in etiolated wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), or Arabidopsis, which lack SafBA. SafBP is most abundant in the coleoptile and scarcest in the leaves, consistent with the distribution of SafBA. SBP1, a cDNA encoding SafBP, was cloned using polymerase chain reaction primers based on purified proteolytic peptides. Extracts of Escherichia coli cells expressing SBP1 have strong [³H]Saf binding, which, like binding to the native maize protein, is competitively inhibited by the safener dichlormid and the herbicides S-ethyl dipropylthiocarbamate, alachlor, and metolachlor. SBP1 is predicted to encode a phenolic O-methyltransferase, but SafBP does not O-methylate catechol or caffeic acid. The acquisition of its encoding gene opens experimental approaches for the evaluation of the role of SafBP in response to the relevant safeners and herbicides.     

Biosynthesis of the Monoterpenes Limonene and Carvone in the Fruit of Caraway : I. Demonstration of Enzyme Activities and Their Changes with Development

The biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway (Carum carvi L.) proceeds from geranyl diphosphate via a three-step pathway. First, geranyl diphosphate is cyclized to (+)-limonene by a monoterpene synthase. Second, this intermediate is stored in the essential oil ducts without further metabolism or is converted by limonene-6-hydroxylase to (+)-trans-carveol. Third, (+)-trans-carveol is oxidized by a dehydrogenase to (+)-carvone. To investigate the regulation of monoterpene formation in caraway, we measured the time course of limonene and carvone accumulation during fruit development and compared it with monoterpene biosynthesis from [U-¹⁴C]Suc and the changes in the activities of the three enzymes. The activities of the enzymes explain the profiles of monoterpene accumulation quite well, with limonene-6-hydroxylase playing a pivotal role in controlling the nature of the end product. In the youngest stages, when limonene-6-hydroxylase is undetectable, only limonene was accumulating in appreciable levels. The appearance of limonene-6-hydroxylase correlates closely with the onset of carvone accumulation. At later stages of fruit development, the activities of all three enzymes declined to low levels. Although this correlates closely with a decrease in monoterpene accumulation, the latter may also be the result of competition with other pathways for substrate.     

Intratrophic predation in simple predator-prey models

Intratrophic predation is a phenomenon not usually considered in mathematical models of biological populations, and yet it may occur in any model where many species are considered as a single model variable. This paper demonstrates how intratrophic predation can be rationally included in a general predator-prey model, and shows that the resulting model has some desirable and intuitively plausible features. A simple asymptotic method is developed in order to investigate how intratrophic predation can affect both the position and stability of the equilibria of a model. The methods can be applied to wide classes of population models, and the conclusions drawn are of practical importance.     

Extinction times and phase transitions for spatially structured closed epidemics

This paper considers the time to extinction for a stochastic epidemic model of SEIR form without replacement of susceptibles. It first shows how previous rigorous results can be heuristically explained in terms of the more transparent dynamics of an approximating deterministic system. The model is then extended to include a host population structured into patches, with weak nearest-neighbour mixing of infection. It is shown, by considering the approximating deterministic system, that the expected time to extinction in a population of n + 1 patches each of size N is of the form a log N + bn, provided that N > N _c where N _c is a critical patch size below which transits are unlikely to occur. This corresponds to the simple decomposition of the time of an epidemic into the time it takes to spread through one patch plus the time it takes to transit to each of n successive patches. Expressions for this threshold and the coefficients of the time to extinction are given in terms of the transmission parameters of infection and the coupling strength between patches. These expressions are compared with numerical results using parameters relevant to a study of phocine distemper virus in North Sea seals, and the agreement is found to be good for large and small N. In the region when N ≈ N _c , where transits may or may not occur, interesting transitional behaviour is seen, leading to a non-monotonicity of the extinction time as a function of N.     

The Interleukin-17 Gene of Herpesvirus Saimiri

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.     

OVX1 and CEA in patients with colon carcinoma, colon polyps and benign colon disorders

The OVX1 tumor marker promises to complement CA125 for detection of early stage ovarian carcinoma. OVX1 has also been shown to be elevated in colon cancer patients. This study is designed to assess serum OVX1 levels in patients with specific stages of colon cancer, colon polyps or other GI disorders. Serum OVX1 and CEA were measured by radioimmunoassay or enzyme immunoassay for 206 patients at the time of colonoscopy or staging for colon carcinoma. In patients with stage I, II, III, or IV colon carcinoma, serum OVX1 was positive in 37%, 48%, 74% and 63%, respectively. Fifty-three percent of patients with colon polyps had elevated OVX1 levels, while OVX1 levels were positive in only 7% of healthy controls. If both OVX1 and CEA were considered, at least one of these markers was elevated in 36%, 60%, 79% or 89% of patients with stage I, II, III or IV colon carcinoma, respectively. The majority of patients with inflammatory bowel disease or diverticulosis also had elevated OVX1 levels. Both markers were positive in 27% of patients with colon carcinoma, and not in any patients with a normal colonoscopy or with a diagnosis of diverticulosis or hemorrhoids. In conclusion, serum OVX1 improves the sensitivity of CEA for detecting colon polyps and colon cancer; however, the use of OVX1 in this setting is hindered by its elevation in non-malignant colonic processes.     

Ski slope vegetation at Snoqualmie Pass, Washington State, USA, and a comparison with ski slope vegetation in temperate coniferous forest zones

Ski slope vegetation at Snoqualmie Pass in Washington State, USA, was surveyed in order to identify community types and to compare it with vegetation development patterns in Japan. Ski slopes in Japan, most of which were constructed after 1960, underwent heavy land recontouring, while those at Snoqualmie Pass were constructed before 1950 with less modification. Three points apply to Japanese ski slope vegetation and differentiate these slopes from those at Snoqualmie Pass: (i) grasslands of introduced species are widespread and persistent; (ii) unvegetated patches are uncommon; and (iii) wetland vegetation has developed. These differences are mainly derived from the intensity of human impact, history of the slope and its scale: namely, ski slopes in Washington are older and larger than those in Japan. Ski slope vegetation in Washington was primarily differentiated by a soil moisture gradient. The large size of Washington ski slopes permitted the inclusion and development of wetland habitats, whereas most ski slopes in Japan are constructed on ridges and do not contain wetlands. Most introduced species in Japan are eliminated soon after seeding. In contrast, the long-term management of ski slopes decreased soil erosion and/or unvegetated patches in Washington and created relatively permanent grasslands composed of introduced species. Tsuga heterophylla and Abies amabilis were found established on the ski slopes in Washington, whereas in Japan the pioneer tree species are shade-intolerant broadleaved species. These differences may be a result of the different disturbance histories of ski slopes in the two countries. In addition, along with the conifers, early successional forbs such as Anaphalis margaritacea and Epilobium angustifolium are well established on Washington ski slopes. Results show that disturbances created by ski slope development greatly affect the vegetation, even on older, less heavily impacted ski slopes.     

The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.