Characterization of na(+) and ca(2+) channels in zebrafish dorsal root ganglion neurons

Background

Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.

Methodology/Principal Findings

We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na⁺ currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I_Na were preferentially expressed in relatively large neurons, while slowly-inactivating I_Na was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I_Na components. Voltage-gated Ca²⁺ currents (I_Ca) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca_V2.2 (N-type) Ca²⁺ channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I_Ca in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.

Conclusions/Significance

Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.     

Cytoplasmic and serum galectin-3 in diagnosis of thyroid malignancies

In order to address whether galectin-3 in the sera and fine needle aspirates serve as a diagnostic marker distinguishing between benign and malignant thyroid nodules, we developed an enzyme-linked immunosorbent assay. We quantified galectin-3 in fine needle aspirates from a series of 118 patients with thyroid nodules and serum galectin-3 from another series of 46 patients, which were compared with final histology after thyroidectomy. Relative galectin-3 value (ng/mg), defined as galectin-3 concentration (ng/ml) divided by total protein concentration (mg/ml) in fine needle aspirates, was significantly higher in papillary carcinoma than in the other thyroid entities. There was no significant difference in serum galectin-3 level among patients with thyroid nodules and healthy individuals. Accordingly, relative galectin-3 value allows a definitive diagnosis of papillary carcinoma independent of cellular morphology, whereas serum galectin-3 does not serve as a marker for papillary carcinoma.     

Porocottus leptosomus sp. nov., from the west coast of Korea, Yellow Sea (Scorpaeniformes: Cottidae)

 A new cottid species, Porocottus leptosomus, is described on the basis of 12 specimens collected from Taean, west coast of Korea, Yellow Sea. The species is distinguished from other species of Porocottus by the following combination of characters: a strongly compressed body, two pairs of branched cirri on the dorsal surface of the head, bases of head cirri smooth, a single cirrus on the dorsal tip of each spine of the first dorsal fin, a single opening of the sixth canaliculus of the infraorbital sensory canal, a long posterior medial canaliculus of the occipital canal with the terminal pore and a few supplementary pores, and melanophores on the isthmus hidden beneath the branchiostegal membrane. Received: January 11, 2001 / Revised: February 7, 2002 / Accepted: March 4, 2002     

Expression of enzymes and oncogene induced after radiotherapy and/or chemotherapy in patients with brain tumors.

The relationship between the degree of the expression of Cu/Zn SOD, GST-pi and bcl-2 in the initial and recurrent tumor tissue after radiotherapy and/or chemotherapy and the cellular heterogeneity obtained from DNA content by image cytometry was investigated. Subjects were 7 patients who had glial tumors which were surgically removed at onset and removed a second time at recurrence. Radiotherapy and chemotherapy were also administered after initial resection. Immunoreactivity for copper/zinc super oxide dismutase (Cu/Zn SOD), GST (glutathione-S-transferase)-pi, and bcl-2 were evaluated from routinely prepared tissue blocks. Tumors were classified into two groups by cytometric analysis of DNA ploidy in the G2M cell cycle phase. One tumor group consisted of single clonal cells in both the initial and recurrent tumors and the other group consisted of tumors with polyclonal cells in the initial and recurrent tumor. In this study, one patient (case 3) with single clonal cell glioblastoma at recurrence did not show high Cu/Zn SOD activity after radiotherapy and chemotherapy but showed a short survival time after recurrence. In three patients (cases 1, 2, 3) with single clonal-cell glioblastoma, the recurrent tumor cells showed high GST-pi immunoreactivity and survival time was short after recurrence. Tumor cells in two patients (cases 5, 7) with single clonal cell anaplastic glioma at recurrence, showed high GST-pi immunoreactivity and had a short survival time after recurrence. In three single clonal glioblastomas (cases 1, 2, 3), the recurrent tumor showed the increased bcl-2 immunoreactivity and showed a short survival time after recurrence. In two patients (case 5, 7) with single clonal cell anaplastic glioma at recurrence, tumor cells showed a high bcl-2 immunoreactivity and these patients showed a short survival time after recurrence. Although the number of subjects is very small, our study shows that the immunoreactivity of bcl-2 and GST-pi in malignant gliomas may be very important factors in radio- and chemosensitivity, and shows that GST-pi is induced by radiation and anti-cancer drugs.     

Specific binding of lectins with the nucleus of the sea urchin embryo and changes in the lectin affinity of the embryonic chromatin during the course of development

(1) Embryonic cells of sea urchins were made permeable by treating them with glycerol solution for the purpose of allowing penetration of macromolecules into the cell. With the use of such permeabilized cells, several kinds of fluorescent dye-labeled lectins were introduced into the cell, and it was found that some lectins showed notable affinity with the nucleus as compared with cytoplasmic structures. (2) Isolated chromatin was incubated with several kinds of fluorescent dye-labeled lectins in vitro, and the amount of lectins bound with the chromatin was measured by fluorometry. By means of this method, the lectin-binding capacity of chromatin was estimated and compared at various stages of development. It was found that lectins could be classified into three groups according to the mode of binding with the chromatin: (a) Extent of binding increased notably at the gastrula stage (Con A and RCA-120); (b) extent of binding showed a temporary decrease at the gastrula stage (TTA); and (c) very low level of binding was maintained throughout all stages, and no particular change was observed at any stage of development (WGA, SBA, and UEA-I). (3) These facts seem to suggest that lectin-binding components are contained in sea urchin chromatin, and that drastic changes occur in these components of chromatin at the stage of gastrulation. It was proposed that the lectin-binding components such as proteoglycans and glycoproteins may play regulatory roles in embryonic chromatin at early stages of development.     

中国脊髓灰质炎Ⅱ型疫苗相关分离株病毒性状的观察

对１９９４年中国分离的１３株脊髓灰质炎Ⅱ型疫苗相关株进行了ＰＣＲ－ＲＦＬＰ分析,发现７株为重组病毒,毒力较疫苗株有回复,在Ⅱ型脊髓灰质炎病毒基因序列上,对于神经毒力有重要影响的第４８１位核苷酸发生突变,另一个被视为重要位点的２９０８位核苷酸无一发生变化,反而在２９０９位核苷发生了高频率的点突变,意味着２９０９位点在中国Ⅱ型疫苗相关株的自然变异中可能起着重要作用。     

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.     

Benchmarking the effects of operating system interference on extreme-scale parallel machines

We investigate operating system noise, which we identify as one of the main reasons for a lack of synchronicity in parallel applications. Using a microbenchmark, we measure the noise on several contemporary platforms and find that, even with a general-purpose operating system, noise can be limited if certain precautions are taken. We then inject artificially generated noise into a massively parallel system and measure its influence on the performance of collective operations. Our experiments indicate that on extreme-scale platforms, the performance is correlated with the largest interruption to the application, even if the probability of such an interruption on a single process is extremely small. We demonstrate that synchronizing the noise can significantly reduce its negative influence.

Analysis of severe acute respiratory syndrome coronavirus structural proteins in virus‐like particle assembly

SARS‐CoV has four major structural proteins: the N, S, M, and E proteins. To investigate the mechanism of SARS‐CoV assembly, we cloned the genes encoding these four proteins into the eukaryotic expression vector pCAGGS and transfected them into 293T cells. When all four expression vectors were co‐transfected VLP formed, as confirmed using electron microscopy. Using a rabbit polyclonal antibody specific to the N protein, N‐protein‐containing particles similar in size to the VLP were also observed by immunoelectron microscopy, indicating that the VLP contained the N protein. Co‐immunoprecipitation analyses demonstrated an interaction between the N and M proteins, suggesting that N protein binds directly to M protein to be incorporated into VLP.