Structural and functional studies of LaIT2, an antimicrobial and insecticidal peptide from Liocheles australasiae.

LaIT2, composed of 59 amino acid residues, is a peptide toxin isolated from the venom of the Yaeyama scorpion, Liocheles australasiae. LaIT2 is toxic to insects but not most mammals. The N- and C-domains of LaIT2 are known to possess antimicrobial and insecticidal activities, respectively. However, the molecular mechanisms are largely unknown because of the lack of a three-dimensional structure of LaIT2. Thus, we elucidated the solution NMR structure of LaIT2. LaIT2 adopts a ß-KTx-like two-domain structure, in which the N- and C-terminal domains form a random coil and an α-ß-ß motif, respectively. Trifluoro ethanol and liposomes titration experiments showed that the unstructured N-domain of LaIT2 has the ability to form an α-helix. The N-terminal helix is amphiphilic, and one side of the helix is positively charged. Measurements of the antimicrobial and insecticidal activities of LaIT2 mutants suggested K15 in the N-domain was found to be responsible for the antimicrobial activities, whereas L53 and L54 in the C-domain were key residues involved in the insecticidal activity. Moreover, K21 in the N-domain is important for both activities. Therefore, two domains are suggested that they work together to show antimicrobial and insecticidal activity.

Overexpression and purification of a toxic peptide LaIT2 from Japanese scorpion, Liocheles australasiae.

In Japan, there are two species of scorpions, Madara scorpion (Isometrus maculatus) and Yaeyama scorpion (Liocheles australasiae), and both of them are living in Yaeyama island. It has been shown that Liocheles australasiae has venom including ß-toxin acting on K+-channels (ß-KTx) (Juichi et al., 2018) [1]. Interestingly, LaIT2, one of the toxins found in the venom of Liocheles australasiae, displays the virulence for insects but almost not for mammals. Until now, molecular mechanism of the functional specificity of LaIT2 is unknown. To clear this issue, we tried to establish the overexpression system of LaIT2 in Rosetta-gami B (DE3) pLysS, which have trxB/gor mutations to induce the disulfide bond formation. In this study, we have succeeded to overexpress the recombinant LaIT2 (rLaIT2) as a thioredoxin (Trx)-tagged protein, and established the purification protocol with Ni2+-NTA column chromatography, enterokinase digestion, and HPLC. We succeeded to obtain approximately 0.5 mg of rLaIT2 from the E. coli cells cultured in 1 L of M9 culture medium. Intramolecular disulfide bonding pattern of rLaIT2 was identified by endopeptidase fragmentation and mass spectrometry. rLaIT2 showed insecticidal activity and antimicrobial activity, and these are almost identical to those of natural LaIT2. 1H-15N HSQC spectrum of 15N-labeled rLaIT2 indicated that the rLaIT2 has a stable conformation.

A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed "gel shifting" appears to be common for histidine-rich proteins but not yet studied in detail. We investigated "gel shifting" in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining "gel shifting" of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3-4 kDa) than apo-Hpn, phenomenon termed "metal gel-shift" demonstrating an intimate link between Ni2+ binding and "gel shifting". To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66±0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact "metal-gel shift" band on SDS-PAGE. In view of presented data metal-binding and "metal-gel shift" models are discussed.

Hg2+-trapping beads: Hg2+-specific recognition through thymine-Hg(II)-thymine base pairing.

Mercury pollution poses a severe threat to human health. To remove Hg(2+) from contaminated water, we synthesized Hg(2+)-trapping beads that include oligo-thymidine functionalities that can form thymine-Hg(II)-thymine base pairs on the solid support. The beads can selectively trap Hg(2+) even in the presence of other metal cations. More interestingly, Hg(2+)-trapping efficiency was higher in the presence of the co-existing cations. Thus, the developed Hg(2+)-trapping beads can capture Hg(2+) without affecting the mineral balance of water so much. The Hg(2+)-trapping beads presented here show promise for removing Hg(2+) from environmental water.

Improved protein overexpression and purification strategies for structural studies of cyanobacterial metal-responsive transcription factor, SmtB from marine Synechococcus sp. PCC 7002.

There are structural and functional differences in SmtB homologs, metal-responsive transcription factors responsible for sensing of excess heavy metal ions in marine and freshwater cyanobacterial strains. The structure of SmtB from freshwater Synechococcus sp. strain PCC 7942 is elucidated with nuclear magnetic resonance (NMR) and crystallography techniques. But knowledge about the functioning of SmtB homologs from marine species is limited till date. To enable NMR spectroscopic studies for investigating structural and functional aspects, modified protocols with higher yields of isotopically labeled SmtB, from marine species like Synechococcus sp. PCC 7002 are essential. In this study, smtB gene was cloned from genome of Synechococcus sp. PCC 7002 and overexpression protocol for recombinant SmtB was standardized in Escherichia coli containing T7 RNA polymerase/promoter system. Further, the protocol for large-scale production, isotope labeling with (15)N, and purification of recombinant SmtB in E. coli BL21(DE3)/pLysS cells was developed. Purified recombinant protein was successfully used for NMR spectroscopy experiments. These results indicate that the overexpression technique now developed is applicable to the structural and functional studies for the proteins being homologous to cyanobacterial SmtB from Synechococcus sp. PCC 7002.

MKRN expression pattern during embryonic and post-embryonic organogenesis in rice (Oryza sativa L. var. Nipponbare).

Rice MKRN is a member of the makorin RING finger protein gene (MKRN) family, which encodes a protein with a characteristic array of zinc-finger motifs conserved in various eukaryotes. Using non-radioactive in situ hybridization, we investigated the spatio-temporal gene expression pattern of rice MKRN during embryogenesis, imbibition, seminal and lateral root development of Oryza sativa L. var. Nipponbare. MKRN expression was ubiquitous during early organogenesis in the embryo along the apical-basal and radial axes. The expression of MKRN decreased during embryonic organ elongation and maturation compared to early embryogenesis, but increased again during imbibition. Tissue-specific and position-dependent MKRN expression was found during embryonic and post-embryonic root and shoot development. Meristematic cells ubiquitously expressed MKRN transcripts, while differentiating cells showed a gradual reduction and termination of MKRN expression. Interestingly, during post-germination MKRN expression was prominent and continued in the metabolically active, differentiated companion cells of the phloem. The differential expression pattern was observed both in the differentiating and differentiated cells. Also, MKRN was expressed in the various developmental stages of the lateral root primordia and the cells surrounding them. Expression of MKRN was also observed after periclinal division of the presumptive pericycle founder cells. The MKRN expression pattern during development of various growth stages suggests an important role of makorin RING finger protein gene (MKRN) in embryonic and post-embryonic organogenesis in both apical-basal and radial developmental axes of rice.

Elongation factor G is a critical target during oxidative damage to the translation system of Escherichia coli.

Elongation factor G (EF-G), a key protein in translational elongation, is known to be particularly susceptible to oxidation in Escherichia coli. However, neither the mechanism of the oxidation of EF-G nor the influence of its oxidation on translation is fully understood. In the present study, we investigated the effects of oxidants on the chemical properties and function of EF-G using a translation system in vitro derived from E. coli. Treatment of EF-G with 0.5 mM H(2)O(2) resulted in the complete loss of translational activity. The inactivation of EF-G by H(2)O(2) was attributable to the oxidation of two specific cysteine residues, namely, Cys(114) and Cys(266), and subsequent formation of an intramolecular disulfide bond. Replacement of Cys(114) by serine rendered EF-G insensitive to oxidation and inactivation by H(2)O(2). Furthermore, generation of the translation system in vitro with the mutated EF-G protected the entire translation system from oxidation, suggesting that EF-G might be a primary target of oxidation within the translation system. Oxidized EF-G was reactivated via reduction of the disulfide bond by thioredoxin, a ubiquitous protein that mediates dithiol-disulfide exchange. Our observations indicate that the translational machinery in E. coli is regulated, in part, by the redox state of EF-G, which might depend on the balance between the supply of reducing power and the degree of oxidative stress.

A molecular insight into Darwin's "plant brain hypothesis" through expression pattern study of the MKRN gene in plant embryo compared with mouse embryo.

MKRN gene family encodes zinc ring finger proteins characterized by a unique array of motifs (C3H, RING and a characteristic cys-his motif) in eukaryotes. To elucidate the function of the MKRN gene and to draw an analogy between plant root apical meristem and animal brain, we compared the gene expression pattern of MKRN in plant seeds with that of mouse embryo. The spatio-temporal expression of MKRN in seeds of pea and rice was performed using non radioactive mRNA in situ hybridization (NRISH) with DIG and BIOTIN labeled probes for pea and rice embryos respectively. Images of MKRN1 expression in e10.5 whole mount mouse embryo, hybridized with DIG labeled probes, were obtained from the Mouse Genome Database (MGD). MKRN transcripts were expressed in the vascular bundle, root apical meristem (RAM) and shoot apical meristem (SAM) in pea and rice embryos. The spatial annotation of the MKRN1 NRISH of whole mount mouse embryo shows prominent localization of MKRN1 in the brain, and its possible expression in spinal cord and the genital ridge. Localization of MKRN in the anterior and posterior ends of pea and rice embryo suggests to the probable role it may have in sculpting the pea and rice plants. The expression of MKRN in RAM may give a molecular insight into the hypothesis that plants have their brains seated in the root. The expression of MKRN is similar in functionally and anatomically analogous regions of plant and animal embryos, including the vascular bundle (spinal cord), the RAM (brain), and SAM (genital ridge) thus paving way for further inter-kingdom comparison studies.

Comparative study of the different mechanisms for zinc ion stress sensing in two cyanobacterial strains, Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803.

In response to an increased level of Zn(2+), Synechococcus sp. PCC 7942 expresses SmtA, a metallothionein-like metal-chelating protein, while Synechocystis sp. PCC 6803 expresses ZiaA, a transporter of Zn(2+). The gene expression of these proteins is regulated by repressor protein, SmtB and ZiaR, respectively. In spite of contributing to different response systems, both repressor proteins belong to the ArsR family and are highly homologous to each other. To understand the different systems responsible for dealing with excess Zn(2+), we examined the cis-elements in the promoter regions of smtA and ziaA, as well as the binding affinities of recombinant SmtB and ZiaR proteins. The operator/promoter region of smtA included two palindromic sequences and that of ziaA included one. Electrophoretic mobility shift assay revealed that SmtB formed four different complexes with the operator/promoter region of smtA, whereas it formed only two different complexes with the corresponding region of ziaA. For ZiaR, the corresponding results were quite the same as those for SmtB. Furthermore, the complex formation between SmtB and operator/promoter regions is inhibited in the presence of Zn(2+) at higher concentrations than 16 µM. On the other hand, the corresponding Zn(2+) concentration is 128 µM. These results demonstrate that the degrees of protein-DNA complex formation between repressor proteins and the operator/promoter regions of regulated genes depend on the structures of the operator/promoter regions, and the effects of Zn(2+) on the dissociation of these complexes are mainly associated with the structures of the repressors.

In-transit intramammary sentinel lymph nodes from malignant melanoma of the trunk.

OBJECTIVE: Our goal was to determine the incidence and outcomes of intramammary in-transit sentinel lymph nodes (IMSLN) from primary malignant melanoma (MM) of the trunk. We hypothesize that regional metastasis to the breast from anterior trunk MM also occurs via the lymphatic system to these intramammary in-transit sentinel lymph nodes. BACKGROUND: MM is the most common solid tumor metastasis to the breast. The mechanism of intramammary (IM) metastasis is generally attributed to hematogenous rather than lymphatic spread. METHODS: We retrospectively reviewed medical records from all patients who underwent selective sentinel lymph node dissection at the UCSF Melanoma Center from 1993 to 2008 after the approval of UCSF Committee on Human Research. Of the 1911 cases, we found 614 patients with primary MM located on the trunk, and queried their medical records for in-transit SLN and SLNs in the breast. Data from preoperative lymphoscintigraphy, intraoperative lymphatic mapping, operative notes, and pathology and clinic notes were gathered. RESULTS: Of the 1911 patients with MM, 169 (8.9%) and 420 (22.0%) had anterior and posterior trunk lesions, respectively, and 25 patients (1.3%) with flank lesions (lateral abdominal wall below the rib cage, above the iliac crest). Of the anterior trunk population, 18 patients had in-transit SLNs. The vast majority of these patients (14 of 18, 77.8%) had in-transit IMSLN. Of patients with posterior trunk melanoma, 27 patients had in-transit nodes with 1 patient having IMSLNs. Of patients with flank melanomas, 3 patients had in-transit nodes with 1 patient having IMSLNs. Interestingly, all patients with IMSLNs had primary lesions located inferior to the breasts. Two of the 16 patients with IMSLNs had micrometastasis to IMSLN; 1 patient died and the other currently is disease free 4 years after initial SLND. Four of the 32 patients with non-IM in-transit nodes had micrometastases to these in-transit nodes. Of all patients with trunk melanomas, 4 patients had micrometastases to axillary SLNs (AxSLNs). Three of the 4 patients with positive AxSLNs also had positive in-transit nodes whereas only half of the patients with positive in-transit SLNs had positive AxSLNs. CONCLUSIONS: IMSLNs exist in the breast. Our results establish an anatomic basis for lymphatic metastasis to the breast from primary cutaneous melanoma mainly from the anterior trunk inferior to the breasts. For anterior trunk melanomas, IMSLNs should not be overlooked during SLND as they may harbor micrometastasis.

Selective sentinel lymph node dissection for melanoma: importance of harvesting nodes with lower radioactive counts without the need for blue dye.

BACKGROUND: Determining how many sentinel lymph nodes (SLNs) should be removed for melanoma is important. The purpose of this study is to determine the frequency at which nodes that are less radioactive than the "hottest" node (which is negative) are positive for melanoma, how low of a radioactivity should warrant harvest, and if isosulfan blue is necessary. METHODS: We reviewed 1,152 melanoma patients who underwent lymphoscintigraphy with technetium, with or without blue dye, and SLN dissection from 1996 to 2008. SLNs with radioactivity ≥10% of the "hottest" SLN, all blue nodes, and all suspicious nodes were removed and analyzed. The miss rate was calculated as the proportion of node positive cases in which the "hottest" SLN was negative. RESULTS: SLNs were identified in 1,520 nodal basins in 1,152 patients. SLN micrometastases were detected in 218 basins (14%) in 204 patients (18%). In 16% of SLN-positive patients (33/204 patients), the positive SLN was found to have a lower radioactive count than the "hottest" SLN, which was negative. In 21 of these cases, the positive SLNs had radioactivity ≤50% of the "hottest" SLN. The 10% rule significantly reduced the miss rate to 2.5% compared with removal of only the "hottest" SLN (miss rate = 16%). Also, blue dye did not significantly decrease the miss rate compared with radiocolloid alone using the 10% rule. CONCLUSIONS: To decrease the miss rate, all SLNs with ≥10% of the ex vivo radioactivity of the "hottest" SLN should be removed and blue dye is not essential.

Intraoperative frozen section analysis of sentinel lymph nodes in breast cancer patients: a meta-analysis and single-institution experience.

BACKGROUND: Accurate intraoperative pathologic examination of sentinel lymph nodes (SLNs) has been an important tool that can reduce the need for reoperations in patients with SLN-positive breast cancer. The objective of the current study was to determine the accuracy of intraoperative frozen section (IFS) of SLNs during breast cancer surgery. METHODS: The authors retrospectively reviewed the records of 326 patients with breast cancer who underwent IF analysis of SLNs at a single institution. Then, they conducted a meta-analysis that included 47 published studies of IFS of SLNs in patients with breast cancer. RESULTS: Hematoxylin and eosin (H&E) staining revealed metastasis in SLNs in 99 patients (30.4%), including 61 patients with macrometastasis (MAM) (>2 mm) (the MAM group) and 38 patients with micrometastasis (Mi) or isolated tumor cell (ITC) deposits (the Mi/ITC group). The overall sensitivity of the institutional series was 60.6% (60 of 99 patients), and overall specificity was 100% (227 of 227 true negatives). The sensitivity of IFS was significantly lower in the Mi/ITC group (28.9%) than in the MAM group (80.3%; P < .0001). According to the meta-analysis of published studies and data from the author's institution (47 studies, for a total of 13,062 patients who underwent SLN dissection with IFS of SLNs), the mean sensitivity was 73%, and the mean specificity was 100%. The mean sensitivity was 94% for the MAM group and 40% for the Mi/ITC group. CONCLUSIONS: IFS of SLNs was more reliable for detecting MAM than for detecting Mi/ITC deposits. It lacked sufficient accuracy to rule out Mi/ITC deposits.

Immunohistochemical localization of apyrase during initial differentiation and germination of pea seeds.

Localization of the 49-kDa apyrase (ATP diphosphohydrolase, EC3.6.1.5; DDBJ/EMBL/GenBank BAB40230) was investigated during early stages of germination of pea (Pisum sativum L. var. Alaska) at the organ, tissue, cellular, and sub-cellular level using light-microscopical immunohistochemistry. Whole mount tissues were immuno-reacted with anti-APY1 serum, pre-immune serum or anti-actin antibody for control. Antigen to the anti-APY1 serum was not detected until 16 h after sowing (26 h after start of imbibition), when the antigen was detected throughout the tissue, especially in the epidermis and cortex. At 35 h after sowing, the younger regions including the root tip and the tip of the stele were more strongly stained than the control. Both, epidermal and cortical cells of the epicotyl and root tip were stained. The stain was mainly localized in the cytoplasm and around nuclei in the apical meristem and the root tip, while vacuoles and cell walls were not stained. At 62 h, there was major staining in the plumule, hook, and elongating regions of the epicotyl and in the region between cotyledons and the epicotyl. After 84 h, lateral root primordia were stained. The pre-immune serum showed virtually no staining while the anti-actin antibody reacted solely with the cytoplasm. Since the antigen to the anti-APY1 serum was primarily found in the cytoplasm and around nuclei in elongating and differentiating tissues and labeling declined in mature tissues, it is suggested that apyrases may play a role in growth and development of tissues, for example, lateral roots.

Prognostic implications of positive nonsentinel lymph nodes removed during selective sentinel lymphadenectomy for breast cancer.

Nonsentinel lymph nodes (SLNs) are commonly removed at the time of selective sentinel lymphadenectomy (SSL). Their predictive value for the rest of the nodal basin is unknown. A retrospective review of 436 breast cancer patients who underwent SSL between 12/97 and 04/03 at a single institution. One-hundred nineteen patients had non-SLNs removed at SSL; eight were positive (6.7%). Positive non-SLNs predicted that SLNs would also be positive (p = 0.008). There was no difference in rates of additional positive nodes found on completion axillary node dissection between the non-SLN and SLN positive patients (p = 0.62). After adjustment for covariates, the presence of positive non-SLNs was not associated with poorer disease free survival (p = 0.24), time to systemic recurrence (p = 0.57), or overall survival (p = 0.70). Positive non-SLNs removed during SSL are not a significant risk factor for additional positive nodes on completion axillary nodal dissection (CALND) or for worse survival than positive SLNs.

NMR-based characterization of a refolding intermediate of beta2-microglobulin labeled using a wheat germ cell-free system.

In patients with dialysis-related amyloidosis, beta2-microglobulin (beta2-m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of beta2-m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non-native trans-Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell-free protein synthesis and NMR techniques. The HSQC spectra of beta2-ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the beta-sheet regions especially the last half of the betaB strand and the first half of the betaE strand, both suggested to be important for amyloidogenicity, may transform beta2-m into an amyloidogenic structure.

TTP at Ser245 phosphorylation by AKT is required for binding to 14-3-3.

Transferrin receptor trafficking protein (TTP) is a key molecule for selective internalization of the transferrin receptor (Tf-R) through endocytic protein complexes. To identify the proteins that directly regulate TTP, we performed a yeast two-hybrid analysis and identified 14-3-3, which can modulate the activation state of target proteins. Subsequent analyses demonstrated that TTP directly binds to multiple 14-3-3 isotypes via its Ser(245) residue (Ser(246) in human) and that these proteins are associated at the plasma membrane. Ser(245) was also found to be a substrate for AKT and the resulting Ser(245) phosphorylation induced the TTP-14-3-3 interaction. Exposure to hydrogen peroxide rapidly enhanced this association in an ovarian cell line. These results suggest that TTP Ser(245) is the principal target for the modulation of this protein via the AKT signalling cascade.

Is it necessary to harvest additional lymph nodes after resection of the most radioactive sentinel lymph node in breast cancer?

BACKGROUND: No consensus exists about the number of sentinel lymph nodes (SLNs) that should be removed based on radioactivity counts in breast cancer, although the "10% rule" is often used. We hypothesized that the node with the highest radioactivity would have the strongest probability of being a positive SLN, and we sought to determine the lowest radioactive count of a node harboring cancer. STUDY DESIGN: We retrospectively studied 332 breast cancer patients who underwent lymphoscintigraphy by injection of technetium 99m-labeled thiosulfate colloid and sentinel lymphadenectomy (SL) between 1997 and 2006, with intraoperative determination of radioactive counts of nodes by a gamma probe. All SLNs were examined by permanent sections consisting of at least 3 levels of 40- to 100-mum intervals for hematoxylin and eosin evaluation, with or without immunohistochemical staining for cytokeratins. RESULTS: Seventy-four percent of patients had more than 1 SLN removed (mean 2.8 per patient); 23.5% had SLN metastasis. Of the node-positive patients, the hottest SLN was positive in 85.9% (67 of 78). Five of the 78 patients (6.4%) with positive nodes had counts less than 10% of those of the hottest node. The lowest radioactive count of a positive SLN was 4.2% of that of the hottest node. Lymphatic mapping based on the 10% rule could greatly improve the false-negative rates compared with removing only the hottest SLN (14.1% versus 6.4%). CONCLUSIONS: Most positive SLNs had the highest radioactivity. Our institutional experience indicates that to obtain an acceptable false-negative rate, nodes should be removed until the 10% rule is met.

Sequence, expression and tissue localization of a gene encoding a makorin RING zinc-finger protein in germinating rice (Oryza sativa L. ssp. Japonica) seeds.

The makorin (MKRN) RING finger protein gene family encodes proteins (makorins) with a characteristic array of zinc-finger motifs and which are present in a wide array of eukaryotes. In the present study, we analyzed the structure and expression of a putative makorin RING finger protein gene in rice (Oryza sativa L. ssp. Japonica cv. Nipponbare). From the analysis of the genomic (AP003543), mRNA (AK120250) and deduced protein (BAD61603) sequences of the putative MKRN gene of rice, obtained from GenBank, we found that it was indeed a bona fide member of the MKRN gene family. The rice MKRN cDNA encoded a protein with four C3H zinc-finger-motifs, one putative Cys-His zinc-finger motif, and one RING zinc-finger motif. The presence of this distinct motif organization and overall amino acid identity clearly indicate that this gene is indeed a true MKRN ortholog. We isolated RNA from embryonic axes of rice seeds at various stages of imbibition and germination and studied the temporal expression profile of MKRN by RT-PCR. This analysis revealed that MKRN transcripts were present at all the time points studied. It was at very low levels in dry seeds, increased slowly during imbibition and germination, and slightly declined in the seedling growth stage. After 6days of germination, an organ-dependent expression pattern of MKRN was observed: highest in roots and moderate in leaves. Similarly to MKRN transcripts, transcripts of cytoskeletal actin and tubulin were also detected in dry embryos, steadily increased during imbibition and germination and leveled off after 24h of germination. We studied the spatial expression profile of MKRN in rice tissues, by using a relatively fast, simple and effective non-radioactive mRNA in situ hybridization (NRISH) technique, which provided the first spatial experimental data that hints at the function of a plant makorin. This analysis revealed that MKRN transcripts were expressed in young plumules, lateral root primordia, leaf primordia, leaves and root tissues at many different stages of germination. The presence of MKRN transcripts in dry seeds, its early induction during germination and its continued spatiotemporal expression during early vegetative growth suggest that MKRN has an important role in germination, leaf and lateral root morphogenesis and overall development in rice.

Impact of multiple lymphatic channel drainage to a single nodal basin on outcomes in melanoma.

OBJECTIVE: To determine the impact of multiple lymphatic channels (MLCs) on outcome in melanoma. DESIGN: Retrospective cohort study. SETTING: Academic tertiary care center. PATIENTS: Of 1198 consecutive selective sentinel lymphadenectomies performed from 1995 to 2000 for primary invasive melanoma, 502 patients were identified with extremity or truncal melanoma that drained to a single nodal basin. Three cohorts were formed based on lymphatic channels (none, single, and multiple). Tumors with drainage to multiple nodal basins as well as all head and neck tumors were excluded. MAIN OUTCOME MEASURES: Multiple variables, including patterns of lymphatic drainage, were analyzed for impact on disease-free and overall survival. RESULTS: Demographics were similar among groups, with a median follow-up of 5.6 years. Univariate analysis revealed MLCs as an independent risk factor for both disease-free (P = .04) and overall survival (P = .003). Multivariate analysis confirmed that tumor depth, sentinel lymph node status, and MLCs were risk factors for both disease-free and overall survival. Kaplan-Meier analysis showed worse survival in the MLCs group. CONCLUSIONS: Our study reveals that MLCs are an independent risk factor for recurrence and mortality in melanoma. Multiple lymphatic channels may facilitate the process of metastasis.

A phase II trial of rosiglitazone in patients with thyroglobulin-positive and radioiodine-negative differentiated thyroid cancer.

BACKGROUND: Rosiglitazone is a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist that has been shown to induce differentiation, cell cycle arrest, and apoptosis in a variety of human cancers including thyroid cancer. METHODS: Ten patients with differentiated thyroid cancer were enrolled in an open-label, phase II trial of oral rosiglitazone treatment (4 mg daily for 1 week, then 8 mg daily for 7 weeks). The levels of PPARgamma receptor mRNA and protein expression were determined in the patient's neoplasm. RESULTS: Of 10 patients, 4 had positive radioiodine scans after rosiglitazone therapy with uptake in the neck in 3 patients and in the pelvis in 1 patient. After treatment, the serum thyroglobulin level decreased in 2 patients, increased in 5 patients, and was stable in 3 patients. No patient developed clinically important toxicity associated with rosiglitazone treatment. We found no relationship in the level of PPARgamma mRNA and protein expression in patients who had radioiodine uptake compared with those who did not. CONCLUSIONS: Our findings suggest that rosiglitazone treatment may induce radioiodine uptake in some patients with thyroglobulin-positive and radioiodine-negative differentiated thyroid cancer. We found no relationship between the expression level of the PPARgamma mRNA and protein in the neoplasm and radioiodine uptake status after rosiglitazone therapy, questioning the potential pathway of effect.