Application of the dual-micropipet technique to the measurement of tumor cell locomotion

The objective of this work was to characterize tumor cell locomotion in response to chemotactic stimulation using a dual-micropipet assay. The assay involves two micropipets. An individual A2058 human melanoma cell was retained, without pressure gradient, in a pipet of approximately 14 micrometers i.d. A solution of type IV collagen, chosen as the chemotactic source, was placed in another pipet (approximately 10 micrometers o.d.) with zero pressure at the pipet tip. The smaller pipet was then inserted into the larger one containing the melanoma cell. The initial chemoattractant concentration (C0) and the distance between the tip of the small pipet and the cell surface (delta) provided a gradient (C0/delta) for tumor cell locomotion toward stimulation. This novel assay provides a direct measure of cell movement: cyclic pseudopod protrusion (Lp) and subsequent cell locomotion (Lc). The influences of different adhesion substrates on cell locomotion were also studied. The peak length in Lp precedes the highest locomotion velocity (dLc/dt) by an apparent lag time. C0/delta influences pseudopod protrusion frequency (fp) and dLc/dt, but not significantly on Lp. Substrate adhesions affect dLc/dt, but apparently not Lp or fp. In conclusion, pseudopod protrusion and substrate adhesion are two necessary but mutually independent factors in tumor cell locomotion. dLc/dt correlates with changes in C0/delta, which is in significant correlation with fp but not Lp.     

The murine homolog (Mph) of human herpesvirus entry protein B (HveB) mediates entry of pseudorabies virus but not herpes simplex virus types 1 and 2

A mouse member of the immunoglobulin superfamily, originally designated the murine poliovirus receptor homolog (Mph), was found to be a receptor for the porcine alphaherpesvirus pseudorabies virus (PRV). This mouse protein, designated here murine herpesvirus entry protein B (mHveB), is most similar to one of three related human alphaherpesvirus receptors, the one designated HveB and also known as poliovirus receptor-related protein 2. Hamster cells resistant to PRV entry became susceptible upon expression of a cDNA encoding mHveB. Anti-mHveB antibody and a soluble protein composed of the mHveB ectodomain inhibited mHveB-dependent PRV entry. Expression of mHveB mRNA was detected in a variety of mouse cell lines, but anti-mHveB antibody inhibited PRV infection in only a subset of these cell lines, indicating that mHveB is the principal mediator of PRV entry into some mouse cell types but not others. Coexpression of mHveB with PRV gD, but not herpes simplex virus type 1 (HSV-1) gD, inhibited entry activity, suggesting that PRV gD may interact directly with mHveB as a ligand that can cause interference. By analogy with HSV-1, envelope-associated PRV gD probably also interacts directly with mHveB during viral entry.     

Defective Glucose Transport Across Brain Tissue Barriers: A Newly Recognized Neurological Syndrome

Impaired glucose transport across brain tissue barriers causes infantile seizures, developmental delay and acquired microcephaly. Since the first report in 1991 (De Vivo et al, NEJM, 1991) 17 patients have been identified with the glucose transporter protein syndrome (GTPS). The diagnostic feature of the syndrome is an unexplained hypoglycorrhachia in the clinical setting of an infantile epileptic encephalopathy. We review our clinical experience by highlighting one illustrative case: a 6-year old girl who presented at age 2 months with infantile seizures and hypoglycorrhachia. The CSF/blood glucose ratio was 0.33. DNA sequencing identified a missense mutation in exon 7 (C1108T). Erythrocyte GLUT1 immunoreactivity was normal. The time course of 3-0-methyl-glucose (3OMG) uptake by erythrocytes of the patient was 46% that of mother and father. The apparent K_m was similar in all cases (2–4 mmol/L), but the apparent V_max in the patient was only 28% that of the parents (500 versus 1,766 fmol/s/10⁶RBC; p < 0.004). In addition, a 3-month trial of oral thioctic acid also benefited the patient and increased the V_max to 935 fmol/s/10⁶ RBC (p < 3 × 10^–7). Uptake of dehydroascorbic acid by erythrocytes of the patient was impaired to the same degree as that of 3OMG (V_max was 38% of that of the mother's), which supports previous observations of GLUT1 being multifunctional. These studies confirm the molecular basis of the GTPS and the multifunctional role of GLUT1. The need for more effective treatment is compelling.     

pH-dependent conformational changes of ferricytochrome c induced by electrode surface microstructure

pH-dependent processes of bovine heart ferricytochrome c have been investigated by electronic absorption and circular dichroism (CD) spectra at functionalized single-wall carbon nanotubes (SWNTs) modified glass carbon electrode (SWNTs/GCE) using a long optical path thin layer cell. These methods enabled the pH-dependent conformational changes arising from the heme structure change to be monitored. The spectra obtained at functionalized SWNTs/GCE reflect electrode surface microstructure-dependent changes for pH-induced protein conformation, pK_a of alkaline transition and structural microenvironment of the ferricytochrome c heme. pH-dependent conformational distribution curves of ferricytochrome c obtained by analysis of in situ CD spectra using singular value decomposition least square (SVDLS) method show that the functionalized SWNTs can retain native conformational stability of ferricytochrome c during alkaline transition.     

Two-step total gene synthesis method

In the post-genomic era, the ability to synthesize any arbitrary DNA sequence is increasingly in demand. A bottleneck in current gene synthesis technologies is the associated cost, due primarily to the high cost of oligonucleotides synthesis and post-synthesis sequencing. In the present paper, an improved method for low-cost gene synthesis that combines dual asymmetrical PCR and overlap extension PCR is presented, which enables any DNA sequence to be synthesized error free. Additionally, the method is easily amenable to automation.     

Comparative study on the modulation of IgE and cytokine production by Phellinus linteus grown on germinated brown Rice, Phellinus Linteus and germinated brown rice in murine splenocytes

We compared the immunomodulating activities in mice of extracts from Phellinus linteus grown on germinated brown rice (PB), Phellinus linteus (PL) alone, and germinated brown rice (BR) alone. The PL, BR and PB-treated mice were administered with the respective extract (2 mg/head/day) by oral gavage for 4 weeks. All extracts markedly decreased the IgE production and allergic responses in serum and splenocytes. PL and PB increased the proportion of CD4(+) but not CD8(+) T cells in splenocytes. Cytokine production was significantly augmented in all treated mice; the concentration of IFN-gamma was greater in the PL, BR and PB mice than in the control group. The concentration of IL-10 was lower in the BR group than in the other groups. These results may be related to the suppression of IgE production. We conclude that PB modulated the immune responses of IgE production and Th1/Th2 cytokine secretion in murine splenocytes.     

Molecular characterization of the tobacco SET domain protein NtSET1 unravels its role in histone methylation, chromatin binding, and segregation

Plants contain a great number of genes encoding a distinctive class of SET domain proteins which harbor a plant-specific N-terminal part together with a C-terminal part showing highest sequence similarity to the catalytic domain of the yeast CLR4, the human SUV39H1 and G9a histone-methyltransferases (HMTases). Here we show that NtSET1, a representative member of this class from tobacco, methylated both K9 and K27 of histone H3 in vitro. Ectopic expression of NtSET1, by an inducible promoter, increased the amount of dimethylated H3K9 and induced chromosome-segregation defects in tobacco BY2 cells. Deletion analyses show that the HMTase activity, the association with specific chromatin regions and with condensed chromosomes, and the cellular effects largely depended on the C-terminal region including the SET domain of the protein. Nevertheless, the N-terminal part of NtSET1 was capable of targeting the green fluorescent protein to interphase chromatin. Finally, we show that NtSET1 bound LHP1, the Arabidopsis homolog of animal heterochromatin protein 1 (HP1), and that LHP1 co-localized with heterochromatin containing high amounts of dimethylated H3K9, suggesting a role for NtSET1 in heterochromatic function. Taken together, our results provide new insights into the molecular and global chromatin-binding activities of this particular class member of plant SET domain proteins.     

Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats

We hypothesized that aging is characterized by a reduced release of nitric oxide (NO) in response to shear stress in resistance vessels. Mesenteric arterioles and arteries of young (6 mo) and aged (24 mo) male Fischer 344 rats were isolated and cannulated. Shear stress (15 dyn/cm(2))-induced dilation was significantly reduced and shear stress (1, 5, 10, and 15 dyn/cm(2))-induced increases in perfusate nitrite were significantly smaller at all shear stress levels in vessels of aged rats. Inhibition of NO synthesis abolished shear stress-induced release of nitrite. Furthermore, shear stress (15 dyn/cm(2))-induced release of nitrate was significantly higher and total nitrite (nitrite plus nitrate) was significantly lower in vessels of aged rats. Tiron or SOD significantly increased nitrite released from vessels of aged rats, but this was still significantly less than that in young rats. Superoxide production was increased and the activity of SOD was decreased in vessels of aged rats. There were no differences in endothelial NO synthase (eNOS) protein and basal activity or in Cu/Zn-SOD and Mn-SOD proteins in vessels of the two groups, but extracellular SOD was significantly reduced in vessels of aged rats. Maximal release of NO induced by shear stress plus ACh (10(-5) M) was comparable in the two groups, but phospho-eNOS in response to shear stress (15 dyn/cm(2)) was significantly reduced in vessels of aged rats. These data suggest that an increased production of superoxide, a reduced activity of SOD, and an impaired shear stress-induced activation of eNOS are the causes of the decreased shear stress-induced release of NO in vessels of aged rats.