Investigation of neutral endopeptidases (EC 3.4.24.11) and of neutral proteinases (EC 3.4.24.4) using a new sensitive two-stage enzymatic reaction

A sensitive two-stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3-carboxypropanoyl-alanyl-alanyl-leucine-4-nitroanilide supplemented with Streptomyces griseus amino-peptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5, 25 degrees C) with a catalytic efficiency (kcat = 1.2 x 10(2) s-1, Km = 0.15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (kcat = 1.2 x 10(3) s-1) but the overall efficiency is diminished by a higher Km (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of Bacillus cereus have been assayed sensitively for their neutral endopeptidases and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.     

Effect of the lipophilic/hydrophilic character of cationic triarylmethane dyes on their selective phototoxicity toward tumor cells.

The observation that enhanced mitochondrial transmembrane potential is a prevalent tumor cell phenotype has provided the conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemo- and photochemotherapy of neoplastic diseases. Because the plasma transmembrane potential is negative on the inner side of the cell and the mitochondrial transmembrane potential is negative on the inner side of this organelle, extensively conjugated cationic molecules (dyes) displaying appropriate structural features are driven electrophoretically through these membranes and tend to accumulate inside energized mitochondria. As a result of the higher mitochondrial transmembrane potential typical of tumor cells, a number of cationic dyes preferentially accrue and are retained for longer periods in the mitochondria of these cells compared to normal cells. This differential in both drug loading and retention brings about the opportunity to attack and destroy tumor cells with a high degree of selectivity. Only a small subset of the cationic dyes known to accumulate in energized mitochondria mediate the destruction of tumor cells with a high degree of selectivity, and the lack of a reliable model to describe the structural determinants of this tumor specificity has prevented mitochondrial targeting from becoming a more reliable therapeutic strategy. We describe here a systematic study of how the molecular structure of closely related cationic triarylmethanes affects the selectivity with which these dyes mediate the photochemical destruction of tumor cells. Based on our observations of how the lipophilic/hydrophilic character of these dyes affects tumor selectivity, we propose a simple model to assist in the design of new drugs tailored specifically for imaging and selective destruction of neoplastic tissue via mitochondrial targeting.     

Macroinvertebrates as bioindicators of water quality in the Mkondoa River,Tanzania, in an agricultural area

The suitability of using macroinvertebrates as bioindicators of stream water quality was tested in the Mkondoa River in an agricultural area at Kilosa, using the rapid bioassessment protocol. The family biotic index (FBI) showed marked variation in water quality along the stream from values ranging from 4.1 to 5.0 in the upstream reaches, indicating good water quality, 5.3 to 5.5 in the mid-reaches and 6.0 to 6.5 in the lower reaches. The water quality index (WQI) indicated that water quality was fair (77 ± 0.98) in the upstream reach of the Mkondoa, marginal (55 ± 0.86) in the midstream reach and poor (33 ± 0.45) in the downstream reach. There were significant relationships between biological oxygen demand and dissolved oxygen and the occurrence of specific taxa, mainly Chironomus and Caenis. Significant changes in macroinvertebrate abundance were mostly related to changes in water quality. As in other parts of the world, macroinvertebrate communities proved to be good biological indicators of water quality and they should be used as bioindicators in long-term monitoring of this river.     

Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.     

Mitochondrial Effects of Triarylmethane Dyes

The mitochondrial effects of submicromolar concentrations of six triarylmethane dyes, withpotential applications in antioncotic photodynamic therapy, were studied. All dyes promotedan inhibition of glutamate or succinate-supported respiration in uncoupled mitochondria, in amanner stimulated photodynamically. No inhibition of N,N,N,N-tetramethyl-p-phenylenediamine(TMPD) supported respiration was observed, indicating that these dyes do not affectmitochondrial complex IV. When mitochondria were energized with TMPD in the absence ofan uncoupler, treatment with victoria blue R, B, or BO, promoted a dissipation of mitochondrialmembrane potential and increase of respiratory rates, compatible with mitochondrialuncoupling. This effect was observed even in the dark, and was not prevented by EGTA, Mg²⁺ orcyclosporin A, suggesting that it is promoted by a direct effect of the dye on inner mitochondrialmembrane permeability to protons. Indeed, victoria blue R, B, and BO promoted swellingof valinomycin-treated mitochondria incubated in a hyposmotic K⁺-acetate-based medium,confirming that these dyes act as classic protonophores such as FCCP. On the other hand, ethylviolet, crystal violet, and malachite green promoted a dissipation of mitochondrial membranepotential, accompanied by mitochondrial swelling, which was prevented by EGTA, Mg²⁺, andcyclosporin A, demonstrating that these drugs induce mitochondrial permeability transition.This mitochondrial permeabilization was followed by respiratory inhibition, attributable tocytochrome c release, and was caused by the oxidation of NAD(P)H promoted by these drugs.     

Werner syndrome protein directly binds to the AAA ATPase p97/VCP in an ATP-dependent fashion

We have previously shown that the Werner syndrome helicase, WRNp, a member of the RecQ helicase family, forms a tight molecular complex with the p97/Valosin containing protein (VCP), a member of the AAA (ATPases associated with diverse cellular activities) family of proteins. This interaction is disrupted by chemical agents that confer DNA damage, suggesting that VCP plays an important role in the signal-dependent release of WRNp from its nucleolar sequestration site. Here, we characterized the structural requirements for interactions between WRNp and VCP and for the nuclear localization of VCP. We discovered that VCP directly binds to the RQC (RecQ conserved) domain of WRNp, which is a highly conserved motif common to the RecQ helicase family. This interaction is ATP-dependent, suggesting that VCP plays a mechanistic role in releasing WRNp from the nucleolus. Immunohistochemical analysis of various VCP domains and mutated proteins expressed in vitro demonstrated that VCP may contain several hierarchical cellular localization motifs within its domain structure.     

Loss of Klotho during melanoma progression leads to increased filamin cleavage, increased Wnt5A expression, and enhanced melanoma cell motility

We have previously shown that Wnt5A-mediated signaling can promote melanoma metastasis. It has been shown that Wnt signaling is antagonized by the protein Klotho, which has been implicated in aging. We show here that in melanoma cells, expressions of Wnt5A and Klotho are inversely correlated. In the presence of recombinant Klotho (rKlotho), we show that Wnt5A internalization and signaling is decreased in high Wnt5A-expressing cells. Moreover, in the presence of rKlotho, we observe an increase in Wnt5A remaining in the medium, coincident with an increase in sialidase activity, and decrease in syndecan expression. These effects can be inhibited using a sialidase inhibitor. In addition to its effects on Wnt5A internalization, we also demonstrate that Klotho decreases melanoma cell invasive potential by a second mechanism that involves the inhibition of calpain and a resultant decrease in filamin cleavage, which we demonstrate is critical for melanoma cell motility.