MT1-MMP Initiates Activation of pro-MMP-2 and Integrin αvβ3 Promotes Maturation of MMP-2 in Breast Carcinoma Cells

We evaluated cellular mechanisms involved in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2), an enzyme implicated in the malignant progression of many tumor types. Membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves the N-terminal prodomain of pro-MMP-2 thus generating the activation intermediate that then matures into the fully active enzyme of MMP-2. Our results provide evidence on how a collaboration between MT1-MMP and integrin αvβ3 promotes more efficient activation and specific, transient docking of the activation intermediate and, further, the mature, active enzyme of MMP-2 at discrete regions of cells. We show that coexpression of MT1-MMP and integrin αvβ3 in MCF7 breast carcinoma cells specifically enhances in trans autocatalytic maturation of MMP-2. The association of MMP-2′s C-terminal hemopexin-like domain with those molecules of integrin αvβ3 which are proximal to MT1-MMP facilitates MMP-2 maturation. Vitronectin, a specific ligand of integrin αvβ3, competitively blocked the integrin-dependent maturation of MMP-2. Immunofluorescence and immunoprecipitation studies supported clustering of MT1-MMP and integrin αvβ3 at discrete regions of the cell surface. Evidently, the identified mechanisms appear to be instrumental to clustering active MMP-2 directly at the invadopodia and invasive front of αvβ3-expressing cells or in their close vicinity, thereby accelerating tumor cell locomotion.     

Unordered arrangement of chromosomes in the nuclei of chicken spermatozoa

The arrangement of chromosomes in the elongated sperm nuclei of chicken was studied using fluorescence in situ hybridization with probes specific for telomeres of all chromosomes, a microchromosome, the long arm of chromosome 6, the large heterochromatic block on the Z-chromosome, and the same heterochromatic block plus subtelomeric sites on macrochromosomes 1–4. The positions of all probes vary from one sperm to another. No order in chromosome arrangement is apparent. It is suggested that large chromosome size and small chromosome number correlate with constant positions of chromosomes and vice versa. Based on the known quantity of repetitive units of the repeat on the Z-chromosome, the degree of compaction of chromatin in the chicken sperm nucleus is estimated as ca 0.7 Mb/μm. As judged from the length of the heterochromatic region of the Z-chromosome at the lampbrush stage, the total length of the Z-chromosome in mature sperm is 2.5–4 times that of the sperm nucleus. Received: 15 December 1997; in revised form: 24 March 1998 / Accepted: 14 April 1998     

The GAIF-Positive Population of Neurons in the Evolution of the Temnocephalida

The nervous system of three species of the Temnocephalida has been studied using the GAIF method (which in small flatworms mostly reveals sensory catecholaminergic neurons). These species represent the evolution from the primitive “turbellarian-like” temnocephalids to the most specialised ones with tentacles and a sucker. The numbers and positions of GAIF-positive neurons are invariant within each species and do not change from hatching to full maturity. A characteristic unpaired neuron contributing to the innervation of the anterior margin of the body is present in all species: such a cell has previously been found only in marine Thalassovortex tyrrhenicus (Dalyellidae) which confirms close relationships between these taxa. Our series of species shows (i) a reduction in number of GAIF-positive perikarya associated with the lateral cords and reduction of GAIF-positive innervation on the ventral side of the body, which is probably related to the loss of ciliary locomotion (the shift to passive hunting and looping locomotion) and (ii) reinforcement of the GAIF-positive innervation of the anterior end of the body which begins to play an important role in capturing the prey. The retention of the medial unpaired neuron and nearly identical sets of GAIF-positive neurons in Diceratocephala boschmai and Craspedella pedum (rather different in morphology) give the first indication (in the Plathelminthes) of persistence of homologous neurons through significant evolutionary transformations of the organs they innervate.     

Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIA^Glc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIA^Glc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIA^Glc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIA^Glc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIA^Glc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]–[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIA^Glc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.     

Maintenance of the induced hypomethylated state of tobacco nuclear repetitive DNA sequences in the course of protoplast and plant regeneration

Previously, we established experimental conditions allowing us to induce hypomethylation of tandem arrays of highly repetitive DNA sequences in the Nicotiana tabacum L. nuclear genome (M. Bezdk et al. 1991, Planta 184, 487–490). In this paper, we demonstrate that loci containing the highly repetitive sequences of the HRS60 family can maintain the induced hypomethylated state through protoplast regeneration, non-differentiated callus growth, and plant regeneration. The hypomethylation, induced with 5-azacytidine and monitored on these sequences, did not substantially alter the capacity of calli to produce plants. It can be concluded that, in contradistinction of multiple copies of transgenes or ectopic genes which are usually recognized as methylation targets, endogenous tandem repeats, such as the HRS60, present at 10⁵ copies in the genome, can escape de-novo methylation.Abbreviation AzaC 5-azacytidine This project was supported by the Grant Agency of the Academy of Sciences of the Czech Republic. We thank Ms. Libue Jedliková, Ms. Hana Suchánková, and Ms. Emilie Koudelková for technical assistance.     

Activities of a Mechanosensitive Ion Channel in anE. Coli Mutant Lacking the Major Lipoprotein

Summary The activity of the mechanosensitive (MS) ion channels in membrane patches, excised fromE. coli spheroplasts, was analyzed using the patch-clamp technique. Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wildtype parent were examined. The MS-channel activities in the wild-type membrane rarely revealed substates at the time resolution used. These channels showed a stretch sensitivity indicated by the IISP (the suction for ane-fold increase in channel open probability) of 4.9 mm Hg suction. The MS-channel activities oflpp included a prominent substate and showed a weaker mechano-sensitivity with an 1/S _p of 10.0 mm Hg. Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS channel in the wild-type membrane under minimal suction, only the larger lysolecithin could activate the MS channel in thelpp membranes. After lysolecithin addition, thelpp membrane became more effective in transmitting the stretch force to the MS channel, as indicated by a steepening of the Boltzmann curve. We discuss one interpretation of these results, in which the major lipoprotein serves as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.     

Getting to the inside of cells using metabolic control analysis

Metabolic control analysis can relate control properties of an intact system to kinetic properties (elasticity coefficients) of the enzymes within that system. The method formulating the former as matrix inverse of the latter is elaborated here for the general case and founded in standard metabolic control theory. Then a method is developed that accomplishes the reverse: it is shown that a matrix containing all elasticity coefficients and information concerning the pathway structure equals the inverse of a matrix containing flux and concentration control coefficients. As a consequence, by measuring the control properties of an intact system, one is able to deduce its in situ pathway structure and enzyme kinetic properties: This solves the ever-present question of whether the kinetic properties of enzymes in their isolated state differ from those under the conditions prevailing in the cell.     

The Product of glnL Is Not Essential for Regulation of Bacterial Nitrogen Assimilation

The glnL product is not necessary for the control of expression of glnA and other nitrogen-regulated genes, but it presumably communicates reduntant information on the availability of ammonia from an ammonia-sensitive system consisting of the products of glnB and glnD to the regulatory products of glnF and glnG.