CXCL12 and C5a trigger cell migration via a PAK1/2-p38alpha MAPK-MAPKAP-K2-HSP27 pathway

Cell migration is critical for many processes, such as angiogenesis, inflammation, development and wound healing, and is also involved in tumour progression and metastasis. Here we show that CXCL12, complement factor 5a (C5a), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF)-BB, which stimulate cell migration, also activate p38alpha MAPK. Pharmacological inhibition of this protein kinase with SB 203580 or BIRB 0796, or the genetic ablation of p38alpha MAPK, blocked cell migration induced by the aforementioned chemo-attractants. Macrophages from mice lacking one or more of the other p38 MAPK isoforms showed normal cell migration in response to C5a. We also show that the activation of p38alpha MAPK in response to CXCL12 requires the p21-activated protein kinases (PAK)-1 and PAK-2. MAPKAP-K2 is a protein kinase that is activated by p38alpha MAPK. Reducing its expression using RNA interference blocked CXCL12-induced HeLa cell migration, while macrophages from mice that do not express MAPKAP-K2 failed to migrate in response to C5a. Moreover, RNA interference against the small heat shock protein 27 (HSP27), a physiological substrate of MAPKAP-K2, blocked the CXCL12-induced cell migration. These results demonstrate a general and essential role of the PAK-p38alpha MAPK-MAPKAP-K2-HSP27 signalling pathway in mediating the effects of chemotactic stimuli on cell migration.     

Encapsulation and suspension of hydrophobic liquids via electro-hydrodynamics

There are situations in which bioactive products of interest in biotechnology turn out to be hydrophobic. To reach high uniform levels of such products in water-based host fluids, such as those existing in many biological environments, one strategy consists on dividing the bioactive product into tiny micrometer (or sub-micrometer) pieces, since these are much more amenable of being uniformly dispersed and stabilized in the host fluid. On the other hand, if the bioactive product must act at specific locations, these micrometer pieces need to be hold in place, an objective that may be achieved by encapsulating them in mats of fibers. Here we demonstrate how these tasks may be accomplished by resorting to the generation and control of electrified coaxial jets of a hydrophilic liquid surrounding the hydrophobic liquid carrying the bioactive substance. When the process is carried out inside a dielectric liquid, double oil/water/oil and simple oil/water emulsions may be formed. On the other hand, when the process runs in air and a biopolymer is added to the hydrophilic liquid, then non woven mats of beaded nanofibers, encapsulating the bioactive product in the beads, are generated.     

Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants

Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner.     

Characterization of gentamicin-resistant Mycoplasma hyorhinis.

A study was conducted to determine if a gentamicin-resistant strain of mycoplasma could be developed for use in validating current mycoplasma detection methods for biologic product harvest cell culture fluid (CCF) containing gentamicin. A strain of gentamicin-resistant Mycoplasma hyorhinis was isolated and characterized. The study showed that this organism was similar to the wild-type strain in all ways examined except gentamicin resistance. Both strains of mycoplasma (the gentamicin resistant and the wild-type) exhibited comparable growth patterns and showed 100% homology based on DNA sequencing and analysis of a 464-bp PCR product. Also, analysis using species-specific antisera identified both strains as M. hyorhinis. Two commonly used lot release mycoplasma detection methods (culture and DNAF) consistently detected mycoplasmas in spiked biologic product harvest CCF containing gentamicin but not in unspiked samples. This study demonstrates the first isolation and characterization of a gentamicin-resistant M. hyorhinis that can be used to validate mycoplasma detection methods for biologic product harvest CCF containing gentamicin.     

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.     

Transglutaminase-mediated macromolecular assembly: production of conjugates for food and pharmaceutical applications

Various strategies have been explored in the last 20 years to modify the functional properties of proteins and, among these, protein/polymer conjugation resulted one of the most successful approaches. Thus, the surface modification of polypeptides of potential industrial interest by covalent attachment of different macromolecules is nowadays regarded as an extremely valuable technique to manipulate protein activities. Protein derivatives with a number of either natural or synthetic polymers, like different polysaccharides or polyethylene glycol, have been obtained by both chemical and enzymatic treatments, and in this context, the crosslinking enzyme transglutaminase is attracting an increasing attention as a simple and safe means for protein processing in vitro. In this short review, we summarized the most significant experimental findings demonstrating that a microbial form of the enzyme is an effective tool to obtain several biopolymer-based conjugates potentially useful for both food and pharmaceutical applications.     

An Ultrastructural Study of Pollen Grains Consumed by Larvae of Osmia Bees (Hymenoptera,Megachilidae)

Pollen samples from provisions and faeces found in the nests of four Osmia species were analyzed by SEM and TEM. The pollen grains studied were Cistus, Sonchus type oleraceus. Primus type dulcis, and Quercus type ilex. The apertures of Cistus and Sonchus pollen stored in the provisions were slightly expanded, and the cytoplasm protruded through them. Conversely, Prunus and Quercus pollen grains showed hardly any signs of such apertural protrusions. Further, the cytoplasm of Cistus and Sonchus pollen (which have thin intines) was almost entirely lacking in the pollen grains recovered from faeces, while in the faecal pollen grains of Prunus and Quercus (with thick intines) the cytoplasm was much less modified. These preliminary results indicate that both the protrusion of the cytoplasm in the provisions and the thickness of the intine may play an important role in the digestion of pollen grains by Osmia bee larvae.     

Male-like females, mimicry and transvestism in butterflies (Lepidoptera: Papilionidae)

Abstract. When a butterfly species has a polymorphic female, with one of the forms closely resembling the male, it is customary to suppose that this form is ancestral, and that the ‘odd’ forms have arisen later. R. I. Vane-Wright, on the other hand, has suggested that in some species the male-like form may be a ‘transvestite’ female, the ancestral form of the female having been strikingly unlike the male. As later-derived forms are usually, but not always, genetically dominant to ancestral forms, we can make some choice between these hypotheses by discovering the dominance relations of the male-like and the ‘odd’ forms of the female. In the mimetic Papilio aegeus the male-like form is shown to be recessive to the ‘odd’ (mimetic) form, as has essentially been the case in all other butterflies so far investigated. Papilio phorcas is now shown to be the exception: the ‘odd’ (non-mimetic) form is recessive to the male-like form. We conclude that usually the male-like form is ancestral, but that P.phorcas may be an authentic example of ‘transvestism’, or the ‘transfer’ of male epigamic colour to the female of the species. The yellow, male-like pattern of the mimetic Papilio dardanus may be dominant or recessive to the mimetic forms according to the genetic background: largely recessive in Madagascar, and southern and western Africa, dominant to most forms in Ethiopia, and probably dominant to one mimetic form but recessive to the others in Kenya. All female dardanus patterns, both mimetic and yellow, are strongly dominant to both female phorcas patterns in P. dardanus × P. phorcas hybrids (P. ‘nandina’). The simplest explanation of this situation is that the male-like pattern of dardanus is ancestral, and that dominance has become locally reversed in Ethiopia. The dominance relations, and the sex- or autosomal-linkage of two forms can be determined without pedigree-breeding, simply by observing a few offspring each from a large number of wild-caught females.     

Southern elephant seal (Mirounga leonina) II. Studies of milk protein fractions by gel electrophoresis

 Milk protein fractions during various stages of lactation in the southern elephant seal Mirounga leonina were analysed. Twelve milk samples were taken from ten females throughout the lactation period during 1990 and 1991 at Stranger Point, King George Island, South Shetland Islands. Milk samples were subjected to polyacrylamide gel electrophoresis (PAGE). Samples from different days of lactation gave similar qualitative electrophoretic patterns. True protein content was significantly higher (P<0.05) at the beginning of lactation, and then remained constant until weaning. Caseins and whey proteins each consisted of several protein entities (four and five distinct bands respectively). Casein constituted only about 30% of the protein nitrogen, the remaining 70% being derived from whey proteins. There was some variation in concentration of casein and whey proteins as a function of time (P<0.0.5). Received: 28 July 1993/Accepted: 25 July 1995