Cytokines and tumor markers in potentially malignant disorders and oral squamous cell carcinoma: a pilot study.

OBJECTIVE: To evaluate serum levels of interleukin (IL)-1, IL-6, IL-8, IL-10, soluble IL-2 receptor (sIL-2R), squamous cell carcinoma antigen (SCCA), tissue polypeptide-specific antigen (TPS) and vascular endothelial growth factor (VEGF) in patients with potentially malignant disorders (PMD), oral squamous cell carcinoma (OSCC), or status-post (SP) OSCC. SUBJECTS AND METHODS: Blood was collected from 47 patients, either controls or diagnosed with PMD, OSCC, or SPOSCC. Levels of cytokines and tumor marker were evaluated by ELISAs. Normal levels were based on previous studies and pathology determined by chi-square and Fisher's exact tests. P ≤ 0.05 was considered statistically significant. RESULTS: Above normal levels of SCCA were found for OSCC and dysplasia patients (33.3% and 11.1%, respectively) and high range of normal (upper 20% of the normal range) for lichen planus, SPOSCC, and dysplasia patients (6.67%, 33.3%, and 22.2%, respectively), differences that approached statistical significance (P = 0.055). No differences were found between groups for other tested markers. A progression was seen for SCCA from high range of normal in SPOSCC to a mixture of high normal and elevated in dysplasia to elevated in active OSCC, suggesting that SCCA may be correlated with cancer progression. CONCLUSION: Higher levels of serum SCCA may serve as a marker for dysplasia and progression to oral carcinogenesis.

Engineered neuronal circuits shaped and interfaced with carbon nanotube microelectrode arrays.

Standard micro-fabrication techniques which were originally developed to fabricate semi-conducting electronic devices were inadvertently found to be adequate for bio-chip fabrication suited for applications such as stimulation and recording from neurons in-vitro as well as in-vivo. However, cell adhesion to conventional micro-chips is poor and chemical treatments are needed to facilitate the interaction between the device surface and the cells. Here we present novel carbon nanotube-based electrode arrays composed of cell-alluring carbon nanotube (CNT) islands. These play a double role of anchoring neurons directly and only onto the electrode sites (with no need for chemical treatments) and facilitating high fidelity electrical interfacing-recording and stimulation. This method presents an important step towards building nano-based neurochips of precisely engineered networks. These neurochips can provide unique platform for studying the activity patterns of ordered networks as well as for testing the effects of network damage and methods of network repair.

Compact self-wiring in cultured neural networks.

We present a novel approach for patterning cultured neural networks in which a particular geometry is achieved via anchoring of cell clusters (tens of cells/each) at specific positions. In addition, compact connections among pairs of clusters occur spontaneously through a single non-adherent straight bundle composed of axons and dendrites. The anchors that stabilize the cell clusters are either poly-D-lysine, a strong adhesive substrate, or carbon nanotubes. Square, triangular and circular structures of connectivity were successfully realized. Monitoring the dynamics of the forming networks in real time revealed that the self-assembly process is mainly driven by the ability of the neuronal cell clusters to move away from each other while continuously stretching a neurite bundle in between. Using the presented technique, we achieved networks with wiring regions which are made exclusively of neuronal processes unbound to the surface. The resulted network patterns are very stable and can be maintained for as long as 11 weeks. The approach can be used to build advanced neuro-chips for bio-sensing applications (e.g. drug and toxin detection) where the structure, stability and reproducibility of the networks are of great relevance.

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera.

The binding of [(3)H]tyrosyl-PBAN28-33NH(2) to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO(3)(-) ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K(d) of 5.73 +/- 1.05 x 10(-6) M and a Bmax of 1.85 +/- 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH(2) and PBAN28-33NuEta(2) with a K(i) of 4.3 +/- 1.1 x 10(-6) M and 4.9 +/- 2.6 x 10(-6) M, respectively.

Inhibition by anti-malarial drugs of haemoglobin denaturation and iron release in acidified red blood cell lysates--a possible mechanism of their anti-malarial effect?

Intraerythrocytic malaria parasites ingest the cytosol of their host cell and digest it inside their acid food vacuoles. Acidified (pH 4-5.5, 37 degrees C) human red blood cell lysates were used to simulate this process, measuring the denaturation of haemoglobin (Hb) and the release of iron, in the absence or presence of exogenous protease. Spontaneous Hb denaturation and appearance of non-heme iron were observed upon lysate acidification, their rates decreasing with increasing pH, and increasing in the presence of protease. Both processes were inhibited by the quinoline-containing anti-malarial drugs (QCDs) chloroquine, quinine, mefloquine and amodiaquine at concentrations well below those expected in the acidic food vacuole of the parasite. Spectrophotometric analysis indicated that chloroquine complexes with heme in acid-denatured haemoglobin. Other weak bases as well as verapamil and diltiazem, known to reverse the resistance of malarial parasites to chloroquine, were without effect indicating that the action of QCDs is specific. Based on our previous results and the present report, we suggest that iron release in acidified lysates is mediated through the formation of ferryl (Fe(IV)) radicals. QCDs possibly complex with this radical, as they do with heme, and prevent its contact with an adjacent heme molecule which is required for ring opening and iron release. These results may suggest that one of the anti-malarial effects of QCDs is to deprive the parasite of an adequate iron supply. Addition of iron to cultures of Plasmodium falciparum was expected to circumvent the deprivation of iron and reduce the anti-malarial effect of QCDs. However, adding iron as penetrating fructose or nitrilotriacetate complexes did not alter the parasite's susceptibility to chloroquine. Ascorbate markedly increased the release of iron in acidified lysates, and this effect was not reduced by chloroquine. Ascorbate was found to decrease parasite susceptibility to chloroquine, suggesting that iron deprivation may be an important factor in the anti-malarial action of QCDs.

Hemoglobin denaturation and iron release in acidified red blood cell lysate--a possible source of iron for intraerythrocytic malaria parasites.

Intraerythrocytic malaria parasites ingest the cytosol of their host cell and digest it inside their acid food vacuoles. Acidified (pH 4-5.5, 37 degrees C) human red blood cell lysates were used to simulate this process, measuring the denaturation of hemoglobin (Hb) and the release of iron, in the absence or presence of exogenous protease. Spontaneous Hb denaturation and appearance of non-heme iron were observed upon lysate acidification, its rate decreasing with increasing pH, and increasing in presence of protease. Although the pH- and proteolysis-dependent release of iron paralleled Hb denaturation, released iron accounted for only a few percent of degraded Hb. Superoxide dismutase, catalase, and various scavengers of oxidative radicals had no effect on either process, consistent with the involvement of Fe(IV) intermediates in iron release from heme. Histidine and imidazole inhibited iron release, probably by binding directly to heme. Ascorbate enhanced iron release considerably but marginally enhanced the denaturation of Hb, suggesting that redox cycling of lysate free iron accelerated further release from heme. These processes could account for the endogenous supply of iron to the malarial parasite.

Covalent modification of the amine transporter with N,N'-dicyclohexylcarbodiimide.

N,N'-Dicyclohexylcarbodiimide (DCC) has been previously shown to inhibit the amine transporter from chromaffin granules [Gasnier, B., Scherman, D., & Henry, J.P. (1985) Biochemistry 24, 3660-3667]. A study of the mechanism of inhibition is presented together with the demonstration of covalent modification of the protein. DCC inhibits binding of R1 (reserpine) and R2 (tetrabenazine) types of ligands to the transporter as well as transport. Ligands of the R2 type, but not those of the R1 type, protect against inhibition of all the reactions by DCC, i.e., accumulation of serotonin, binding if reserpine (R1 ligand), and binding of ketanserine (R2 ligand). The ability of a given R2 ligand to protect the transporter correlates well with its binding constant. Water-soluble carbodiimides, such as 1-ethyl-3-[3-(diethylamino)propyl]carbodiimide (EDC), do not have any effect on the catalytic activity of the transporter. A fluorescent hydrophobic analogue of DCC, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), inhibits at about the same concentration range as DCC. [14C]DCC labels several polypeptides in the chromaffin granule membranes. Labeling of a polypeptide with an apparent Mr of 80K is inhibited in the presence of R2 ligands. The labeled polypeptide copurifies with the recently identified and isolated transporter [Stern-Bach, Y., Greenberg-Ofrath, N., Flechner, I., & Schuldiner, S. (1990) J. Biol. Chem. 256, 3961-3966].