Formation of tannin-albumin nano-particles at neutral pH as measured by light scattering techniques

Aggregation phenomena of tannin with bovine serum albumin were investigated by light scattering techniques including photon correlation spectroscopy and Rayleigh scattering. Tannin and albumin formed particles with diameters less than 1 microm at neutral pH. As revealed by this study, light scattering methods are useful in investigating aggregation phenomena of biomolecules and in directly quantifying tannin content.     

Peroxisome proliferator activated receptor alpha regulates a male-specific cytochrome P450 in mouse liver

We set out to find if the strain-specific, male-specific hepatic expression of Cyp4a protein in mouse was due to expression of Cyp4a12 and to understand the genetic basis for reported differences in expression. 12-Lauric acid hydroxylase (LAH) activity was found to show higher levels in male ddY, but not C57Bl/6, mouse liver microsomes. The expression of Cyp4a12 mRNA was studied using RNAase protection assays in male and female liver and kidney of nine mouse strains. Cyp4a12 was found to be highly expressed in male liver and kidney, but at much lower levels in female liver and kidney, in all strains studied. Western blotting with an antibody specific for Cyp4a12 confirmed that Cyp4a12 was expressed in a male specific fashion in C57Bl/6 mouse liver. RNAase protection analysis for Cyp4a10 and 14 in ddY mice revealed that neither of these genes showed male-specific expression. To further investigate genetic factors that control male-specific Cyp4a12 expression, PPARalpha+/+ and -/- mice were studied, showing that total P450 and 12-LAH activity was male-specific in +/+, but not -/- mice. RNAase protection assays were used to confirm that Cyp4a12 was lower in -/- mice. However, the male-specific Slp and MUP-1 genes retained hepatic male-specific levels of expression in +/+ and -/- mice, showing that the decrease in Cyp4a12 was not a general effect on male-specific expression. Thus, PPARalpha has a specific effect on constitutive expression of Cyp4a12.     

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates "suicide gene" expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.     

Insulin-like growth factor 1 stimulates KCl cotransport, which is necessary for invasion and proliferation of cervical cancer and ovarian cancer cells

The mechanisms by which insulin-like growth factor 1 (IGF-1) cooperates with membrane ion transport system to modulate epithelial cell motility and proliferation remain poorly understood. Here, we investigated the role of electroneutral KCl cotransport (KCC), in IGF-1-dependent invasiveness and proliferation of cervical and ovarian cancer cells. IGF-1 increased KCC activity and mRNA expression in a dose- and time-dependent manner in parallel with the enhancement of regulatory volume decrease. IGF-1 treatment triggers phosphatidylinositol 3-kinase and mitogen-activated protein kinase cascades leading to the activation of Akt and extracellular signal-regulated kinase1/2 (Erk1/2), respectively. The activated Erk1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways are differentially required for IGF-1-stimulated biosyn-thesis of KCC polypeptides. Specific reduction of Erk1/2 protein levels with small interference RNA abolishes IGF-1-stimulated KCC activity. Pharmacological inhibition and genetic modification of KCC activity demonstrate that KCC is necessary for IGF-1-induced cancer cell invasiveness and proliferation. IGF-1 and KCC colocalize in the surgical specimens of cervical cancer (n = 28) and ovarian cancer (n = 35), suggesting autocrine or paracrine IGF-1 stimulation of KCC production. Taken together, our results indicate that KCC activation by IGF-1 plays an important role in IGF-1 signaling to promote growth and spread of gynecological cancers.     

Plasmodium ookinete-secreted proteins secreted through a common micronemal pathway are targets of blocking malaria transmission

The mosquito midgut ookinete stage of the malaria parasite, Plasmodium, possesses microneme secretory organelles that mediate locomotion and midgut wall egress to establish sporogonic stages and subsequent transmission. The purpose of this study was 2-fold: 1) to determine whether there exists a single micronemal population with respect to soluble and membrane-associated secreted proteins; and 2) to evaluate the ookinete micronemal proteins chitinase (PgCHT1), circumsporozoite and TRAP-related protein (CTRP), and von Willebrand factor A domain-related protein (WARP) as immunological targets eliciting sera-blocking malaria parasite infectivity to mosquitoes. Indirect immunofluorescence localization studies in Plasmodium gallinaceum using specific antisera showed that all three proteins are distributed intracellularly with a similar granular cytoplasmic appearance and with focal concentration of PgCHT1 and PgCTRP, but not PgWARP, at the ookinete apical end. Immunogold double-labeling electron microscopy, using antisera against the membrane-associated protein CTRP and the soluble WARP, showed that these two proteins co-localized to the same micronemal population. Within the microneme CTRP was associated peripherally at the microneme membrane, whereas PgCHT1 and WARP were diffuse within the micronemal lumen. Sera produced against Plasmodium falciparum WARP significantly reduced the infectivity of P. gallinaceum to Aedes aegypti and P. falciparum to Anopheles mosquitoes. Antisera against PgCTRP and PgCHT1 also significantly reduced the infectivity of P. gallinaceum for A. aegypti. These results support the concept that ookinete micronemal proteins may constitute a general class of malaria transmission-blocking vaccine candidates.     

Effect of the posterior cruciate ligament on posterior stability of the knee in high flexion

Most biomechanical studies of the knee have focused on knee flexion angles between 0 degrees and 120 degrees. The posterior cruciate ligament (PCL) has been shown to constrain posterior laxity of the knee in this range of flexion. However, little is known about PCL function in higher flexion angles (greater than 120 degrees ). This in vitro study examined knee kinematics before and after cutting the PCL at high flexion under a posterior tibial load and various muscle loads. The results demonstrated that although the PCL plays an important role in constraining posterior tibial translation at low flexion angles, the PCL had little effect in constraining tibial translation at 150 degrees of flexion under the applied loads.     

A standardized kinesin nomenclature

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.     

Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex

Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation of actin polymerization by the Arp2/3 complex. We demonstrate that L. monocytogenes motility can be separated into an Arp2/3-dependent nucleation phase and an Arp2/3-independent elongation phase. Elongation-based propulsion requires a unique set of biochemical factors in addition to those required for Arp2/3-dependent motility. We isolated fascin from brain extracts as the only soluble factor required in addition to actin during the elongation phase for this type of movement. The nucleation reaction assembles a comet tail of branched actin filaments directly behind the bacterium. The elongation-based reaction generates a hollow cylinder of parallel bundles that attach along the sides of the bacterium. Bacteria move faster in the elongation reaction than in the presence of Arp2/3, and the rate is limited by the concentration of G-actin. The biochemical and structural differences between the two motility reactions imply that each operates through distinct biochemical and biophysical mechanisms.     

Down-regulation of p21WAF1 promotes apoptosis in senescent human fibroblasts: involvement of retinoblastoma protein phosphorylation and delay of cellular aging

It has been suggested that genes which exercise checkpoint control during cell cycle traverse are equally important to the process of apoptotic cell death. In this study, we show that the key cell cycle regulatory gene p21(WAF1) is also involved in the execution of apoptosis. p21(WAF1) expression was down-regulated during NaBu-induced apoptosis of senescent normal diploid human 2BS fibroblasts. Conversely, when p21(WAF1) expression was actively suppressed in 2BS cells by a stably transfected antisense p21(WAF1) construct, apoptosis was accelerated and senescence was delayed, as shown by several markers of cell aging. Down-regulation of p21(WAF1) by antisense caused an increase in the phosphorylation and inactivation of pRb. Phosphorylation of pRb was further enhanced upon induction of apoptosis by NaBu. Our results suggest that p21(WAF1), acting through the phosphorylation of pRb, regulates whether 2BS cells cease to proliferate and become senescent but resistant to apoptosis, or whether they accelerate proliferation while becoming more susceptible to apoptotic stimuli.     

Improving crop salt tolerance

Salinity is an ever-present threat to crop yields, especially in countries where irrigation is an essential aid to agriculture. Although the tolerance of saline conditions by plants is variable, crop species are generally intolerant of one-third of the concentration of salts found in seawater. Attempts to improve the salt tolerance of crops through conventional breeding programmes have met with very limited success, due to the complexity of the trait: salt tolerance is complex genetically and physiologically. Tolerance often shows the characteristics of a multigenic trait, with quantitative trait loci (QTLs) associated with tolerance identified in barley, citrus, rice, and tomato and with ion transport under saline conditions in barley, citrus and rice. Physiologically salt tolerance is also complex, with halophytes and less tolerant plants showing a wide range of adaptations. Attempts to enhance tolerance have involved conventional breeding programmes, the use of in vitro selection, pooling physiological traits, interspecific hybridization, using halophytes as alternative crops, the use of marker-aided selection, and the use of transgenic plants. It is surprising that, in spite of the complexity of salt tolerance, there are commonly claims in the literature that the transfer of a single or a few genes can increase the tolerance of plants to saline conditions. Evaluation of such claims reveals that, of the 68 papers produced between 1993 and early 2003, only 19 report quantitative estimates of plant growth. Of these, four papers contain quantitative data on the response of transformants and wild-type of six species without and with salinity applied in an appropriate manner. About half of all the papers report data on experiments conducted under conditions where there is little or no transpiration: such experiments may provide insights into components of tolerance, but are not grounds for claims of enhanced tolerance at the whole plant level. Whether enhanced tolerance, where properly established, is due to the chance alteration of a factor that is limiting in a complex chain or an effect on signalling remains to be elucidated. After ten years of research using transgenic plants to alter salt tolerance, the value of this approach has yet to be established in the field.