Targeting CXCL5 in Pancreatic Cancer Cells Inhibits Cancer Xenograft Growth by Reducing Proliferation and Inhibiting EMT Progression.

BACKGROUND: Pancreatic cancer (PC) is the most lethal malignant tumor, with average survival period of about 10 months. C-X-C ligand 5 (CXCL5), an important chemokine for immune cell accumulation in tumor tissues, has been reported to be involved in a variety of human cancers. However, the exact role of CXCL5 in PC progression has not been well defined. METHODS: The expression of CXCL5 in PC was analyzed based on online databases and clinical specimens immunohistochemical staining, and Western blotting of CXCL5 in PC cell lines and patient samples. The correlation between CXCL5 expression and prognosis in PC was explored. The role of CXCL5 in PC was investigated through in vitro and in vivo experiments. RESULTS: The expression of CXCL5 was significantly increased in PC tissues compared with that in pancreas tissues, and CXCL5 high expression predicts poor prognosis in PC patients. Further analyses demonstrated that overexpression of CXCL5 in PC cells was positively related to higher proliferation rate, higher migration ability, and higher EMT markers including SNAI2 and TWIST1 of tumor cells in vitro. Consistently, the knockdown of CXCL5 in PC cells harmed the proliferation rate, migration ability, and expression of EMT indexes of tumor cells in vitro. Importantly, knockdown of CXCL5 inhibited the growth of xenograft tumors in vivo. CONCLUSION: CXCL5 high expression predicts poor prognosis in PC patients. CXCL5 promotes PC cell growth and EMT process. Inhibition of CXCL5 may be a potential therapeutic approach for PC.

Architectural effects regulate resource allocation within the inflorescences with nonlinear blooming patterns.

PREMISE: Spatial and temporal resource allocations within inflorescences have been well-studied in many plants based on flowering sequence or floral position. However, there had been few attempts to investigate architectural effects and resource competition in species where the blooming pattern does not follow a linear positional pattern within the inflorescence. Moreover, most flowering plants show female-biased sex allocation in early or basal flowers, but it is unclear in species with inherent and changeless ovule production. METHODS: We investigated intra-inflorescence variation in reproductive traits of Salvia przewalskii, a perennial herb with 4-ouvle ovary flowers and flowering sequence-floral position decoupled inflorescences. To detect the effects of resource competition and architectural effects on reproductive success, we manipulated inflorescence (removed floral buds by position and flowering sequence) and pollination (opened and supplemented pollination). RESULTS: Pollen production and dry mass deceased from bottom to top flowers but did not significantly differ following flowering sequence, resulting in male-biased sex allocation in basal flowers. The seed production, fruit set, and bud development exhibited significant declining trends from proximal to distal positions regardless of the thinning and pollen treatments. Meanwhile, the seed production, fruit set, and bud development success did not significant differ when thinning was conducted according to flowering sequence. CONCLUSIONS: Architectural effects plays a crucial role in resource allocation within decoupled flowering inflorescences. Moreover, our results highlighted that inherent floral traits such as changeless ovule production, may modify architectural effects on sex allocation.

Speciation along the elevation gradient: Divergence of Roscoea species within the south slope of the Himalayas.

The Himalayas with dramatic elevation gradient is one of the global biodiversity hotspots. Although origin of biodiversity of the Himalayas is of great concern, the speciation process within the Himalayas is poorly known. Roscoea within the Himalayas serve as a good model system to test the speciation process along an elevation gradient. 32,375 unlinked SNPs were used to reconstruct phylogenetic relationships and introgression analyses in D-statistics and Fastsimicoal2. Species distribution modeling (SDM) was used to simulate habitat shift of Roscoea species during climate changes. Phylogeny suggested that the speciation order, except R. capitata, was from highland to lowland. D-statistics analyses suggested significant bidirectional ancient introgression between elevation-neighboring clades but no introgression between R. capitata and othern clades and no introgression among extant species. Fastsimicoal2 suggested interspecific introgressions were asymmetric. SDM predicted that habitats of Roscoea shifted to low elevation during cooling age. These results suggested that the sudden uplift of the Himalayas likely promoted speciation by vicariance, and climate cooling drove species divergence towards lower elevation. This study provides explanations for the origin of biodiversity within the Himalayas, and an insight to understand speciation along elevation in the mountainous regions.

Functional implications of the specialized staminal appendages in alpine ginger (Roscoea spp.: Zingiberaceae).

Floral organs are widely believed to enhance the pollination and reproductive success of angiosperms. However, the functional implication of some floral structures is still unknown. In this study, we explored the functional role of staminal appendages on male and female reproductive success of Himalayan Roscoea spp. and tested if their function differed between species with biotic pollination and autonomous selfing. Phenotypic manipulation is a powerful approach to test the functional effect of a particular trait on plant fitness. We compared various proxies of pollination success between intact flowers and flowers with manually excised staminal appendages. We found that the rate of visitation did not differ between intact and manipulated flowers. Our results revealed that in outcrossing Roscoea spp., the staminal appendages act as triggering devices to facilitate pollen release and deposition and also to manipulate the foraging position of pollinators to ensure both male and female reproductive success. In contrast, in autonomously selfing Roscoea spp., the removal of staminal appendages did not affect any aspect of pollination processes. Our results suggest that the staminal appendages are an integral component of outcrossing in Roscoea spp. and are maintained by selection pressure through both male and female reproductive success. This study provides important insights on how variation in breeding systems can provoke changes in the structure and function of floral organs among congeners.

Water mediates fertilization in a terrestrial flowering plant.

Water-mediated fertilization is ubiquitous in early land plants. This ancestral mode of fertilization has, however, generally been considered to have been lost during the evolutionary history of terrestrial flowering plants. We investigated reproductive mechanisms in the subtropical ginger Cautleya gracilis (Zingiberaceae), which has two pollen conditions - granular and filiform masses - depending on external conditions. We tested whether rain transformed granular pollen into filiform masses and whether this then promoted pollen-tube growth and fertilization of ovules. Using experimental manipulations in the field we investigated the contribution of water-mediated fertilization to seed production. Rain caused granular pollen to form filiform masses of germinating pollen tubes, which transported sperm to ovules, resulting in fertilization and seed set. Flowers exposed to rain produced significantly more seeds than those protected from the rain, which retained granular pollen. Insect pollination made only a limited contribution to seed set because rainy conditions limited pollinator service. Our results reveal a previously undescribed fertilization mechanism in flowering plants involving water-mediated fertilization stimulated by rain. Water-mediated fertilization is likely to be adaptive in the subtropical monsoon environments in which C. gracilis occurs by ensuring reproductive assurance when persistent rain prevents insect-mediated pollination.

Delivery System Targeting Hemagglutinin of Influenza Virus A to Facilitate Antisense-Based Anti-H1N1 Therapy.

Antisense oligonucleotides (ODNs) are therapeutic molecules that hybridize to complementary target mRNA sequences. To further overcome the poor cellular uptake of ODNs, we proposed a novel strategy to deliver ODNs by conjugating the anti-influenza A virus (IAV) ODN with a peptide showing high affinity to the hemagglutinin (HA) on the surface of IAV particles or the IAV-infected host cells. The HA-specific binding peptides were selected by phage display, and the individual binding clones are characterized by DNA sequencing, and the selected phage was further assayed by enzyme-linked immunosorbent assay. The final selected HA-binding peptide, SHGRITFAYFAN, was conjugated to an anti-IAV ODN. The delivery efficiency and the anti-IAV effects of the conjugated molecule were evaluated in a cell-culture and a mouse-infection model. The conjugated molecule was successfully delivered into IAV-infected host cells more efficiently than the anti-IAV ODN in vitro and in vivo. Furthermore, the conjugated molecule protected 80% of the mice from lethal challenge and inhibited the plaque count by 75% compared to the unconjugated molecule (60% and 40%). These findings demonstrate that the delivery of antisense oligodeoxynucleotides to infected tissues by a virus-binding peptide-mediated system is a potential therapeutic strategy against IAV.

Coevolutionary elaboration of pollination-related traits in an alpine ginger (Roscoea purpurea) and a tabanid fly in the Nepalese Himalayas.

Geographical variation in the interacting traits of plant-pollinator mutualism can lead to local adaptive differentiation. We tested Darwin's hypothesis of reciprocal selection as a key driving force for the evolution of floral traits of an alpine ginger (Roscoea purpurea) and proboscis length of a tabanid fly (Philoliche longirostris). We documented the pattern of trait variation in R. purpurea and P. longirostris across five populations. At each site, we quantified pollinator-mediated selection on floral display area, inflorescence height and corolla length of R. purpurea by comparing selection gradients for flowers exposed to natural pollination and to supplemental hand pollination. Reciprocal selection between plant and fly was examined at two sites via the relationship between proboscis length and nectar consumption (fly benefit) and corolla length and pollen deposition (plant benefit). Local corolla tube length was correlated with local fly proboscis length among the five sites. We found strong linear selection imposed by pollinators on corolla tube length at all sites, but there was no consistent relationship of fitness to inflorescence height or floral display area. Selection between corolla length and proboscis length was reciprocal at the two experimental sites examined. The geographical pattern of trait variation and the evidence of selection is consistent with a mosaic of local, species-specific reciprocal selection acting as the major driving force for the evolution of corolla length of R. purpurea and proboscis length of P. longirostris.

Biological Evaluation of Lysionotin: a Novel Inhibitor of 5-Lipoxygenase for Anti-glioma.

OBJECTIVE: To explore the potential mechanism of lysionotin in treating glioma. METHODS: First, target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin. The binding between 5-lipoxygenase (5-LO) and lysionotin was detected by surface plasmon resonance (SPR) and molecular docking, and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis, respectively. Furthermore, the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein expression, intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot, flow cytometry and fluorescence labeling, respectively. RESULTS: Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO, a key enzyme involved in the arachidonic acid metabolism pathway, to inhibit the proliferation of glioma. Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds, forming bonds with His600, Gln557, Asn554, and His372. SPR analysis further confirmed the interaction between 5-LO and lysionotin. Furthermore, enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50% inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58 µmol/L, respectively, and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid (P<0.05), and moreover, the LC-MS/MS results indicated that lysionotin can enter glioma cells well (P<0.01) and inhibit their proliferation. Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO (P<0.05) and downstream leukotriene B4 receptor (P<0.01). In addition, the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton, as determined by flow cytometry and fluorescence labeling. CONCLUSION: Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway.

Associations between high levels of Notch1 expression and high invasion and poor overall survival in hepatocellular carcinoma.

Although Notch1 expression has been associated with progression or prognosis in various tumors, the role of Notch1 in hepatocellular carcinoma (HCC) remains unknown. This study sought to investigate the clinicopathological and prognostic relevance of Notch1 expression in HCC as well as the underlying mechanisms responsible. HCC tissues were stained with an anti-Notch1 antibody. The invasion capacities of cells were measured using Transwell cell culture chambers. Reverse transcription PCR and/or western blot were used to evaluate the expression levels of Notch1, matrix metalloproteinase (MMP)-2, and MMP-9. Notch1 expression was downregulated by RNA interference. The activity of MMP-2/MMP-9 was quantified by enzyme-linked immunosorbent assay, and cellular apoptosis was analyzed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Notch1 expression was mainly localized within the cytoplasm and at the cell membrane. High Notch1 expression correlated with tumor size, tumor grade, metastasis, venous invasion, and American Joint Committee on Cancer TNM stage (P < 0.05), and patients with high levels of Notch1 expression were at a significantly increased risk for shortened survival time (P < 0.05). In vitro, the downregulation of Notch1 expression decreased the invasion capacity of HCC cells via the regulation of MMP-2 and MMP-9. The results of the MTT assay showed that downregulation of Notch1 did not affect HCC cell viability. Notch1 may represent a novel candidate marker for patient prognosis as well a molecular target for HCC therapy.

The down-regulation of Notch1 inhibits the invasion and migration of hepatocellular carcinoma cells by inactivating the cyclooxygenase-2/Snail/E-cadherin pathway in vitro.

BACKGROUND: The Notch signaling pathway plays an important role in cancer, but the mechanism by which Notch1 participates in invasion and migration of hepatocellular carcinoma (HCC) cells is unclear. AIMS: Our purpose is to confirm the anti-invasion and anti-migration effects of the down-regulation of Notch1 in HCC cells. METHODS: The invasion and migration capacities of HCC cells were detected with Transwell cell culture chambers. The expressions of Notch1, Notch1 intracellular domain (N1ICD), E-cadherin, Snail, and cyclooxygenase-2 (COX-2) were analyzed by RT-PCR and/or western blotting. Notch1 and Snail were down-regulated by RNA interference, and COX-2 was inhibited by NS-398. Cell apoptosis was analyzed by MTT and flow cytometry. RESULTS: In HCC cells, Snail, Notch1, and COX-2 were up-regulated, and E-cadherin was down-regulated in mRNA and/or protein levels. The down-regulation of Snail or Notch1 or the inhibition of COX-2, respectively, can increase the mRNA and protein expressions of E-cadherin and decrease the invasion and migration capabilities of HCC cell. Down-regulated Notch1 or inhibited COX-2 can reduce the mRNA and protein expressions of Snail. The down-regulation of Notch1 can also reduce the protein expression of COX-2. However, exogenous PGE2 can reverse the role of down-regulated Notch1. The results of MTT and flow cytometry showed that down-regulated Notch1 did not affect HCC cell viability. CONCLUSIONS: Down-regulated Notch1 may be an effective approach to inactivating Snail/E-cadherin by regulating COX-2, which results in inhibiting the invasion and migration of HCC cells. The inhibitory effects of down-regulated Notch1 on cell invasion and migration were independent of apoptosis.

MicroRNA-10b promotes migration and invasion through CADM1 in human hepatocellular carcinoma cells.

MicroRNA-10b (miR-10b) was recently reported to be dysregulated in some types of cancer and to play a role in invasion and metastasis. However, effects and potential mechanisms of action of miR-10b in the metastasis of hepatocellular carcinoma (HCC) have not been explored. In this study, we confirmed that miR-10b is highly expressed in metastatic HCC tissues and in metastatic HCC cell lines by qRT-PCR. Moreover, patients with higher miR-10b expression had significantly poorer overall survival, and high miR-10b expression was an independent predictor of poor prognosis. Inhibition of miR-10b reduced cell migration and invasion in MHCC97H cells, whereas over-expression of miR-10b in HepG2 cells increased cell migration and invasion. Bioinformatics and luciferase reporter assays revealed that miR-10b binds the 3'-UTR of CADM1 mRNA and represses its translation. Western blot and qRT-PCR showed that CADM1 is inhibited by miR-10b over-expression. Silencing of CADM1 resulted in substantially increased cell motility and invasion similar to that observed with over-expression of miR-10b in HepG2 cells. These results suggest that miR-10b may positively regulate the invasion and metastasis of HCC through targeting CADM1.

Stigmatic fluid aids self-pollination in Roscoea debilis (Zingiberaceae): a new delayed selfing mechanism.

BACKGROUND AND AIMS: Delayed selfing is the predominant mode of autonomous self-pollination in flowering plants. However, few delayed selfing mechanisms have been documented. This research aims to explore a new delayed selfing mechanism induced by stigmatic fluid in Roscoea debilis, a small perennial ginger. METHODS: Floral biology and flower visitors were surveyed. The capacity of autonomous selfing was evaluated by pollinator exclusion. The timing of autonomous selfing was estimated by emasculation at different flowering stages. The number of seeds produced from insect-pollination was assessed by emasculation and exposure to pollinators in the natural population. The breeding system was also tested by pollination manipulations. KEY RESULTS: Autonomous self-pollination occurred after flowers wilted. The stigmatic fluid formed a globule on the stigma on the third day of flowering. The enlarged globule seeped into the nearby pollen grains on the fourth flowering day, thus inducing pollen germination. Pollen tubes then elongated and penetrated the stigma. Hand-selfed flowers produced as many seeds as hand-crossed flowers. There was no significant difference in seed production between pollinator-excluded flowers and hand-selfed flowers. When emasculated flowers were exposed to pollinators, they produced significantly fewer seeds than intact flowers. Visits by effective pollinators were rare. CONCLUSIONS: This study describes a new form of delayed autonomous self-pollination. As the predominant mechanism of sexual reproduction in R. debilis, delayed self-pollination ensures reproduction when pollinators are scarce.

Morphological and molecular evidence for natural hybridization in sympatric population of Roscoea humeana and R. cautleoides (Zingiberaceae).

The sympatric occurrence of some species in Roscoea is very common, but little information is available on natural hybridization. However, some intermediate individuals were found on the sympatric population of Roscoea humeana and R. cautleoides at Ganhaizi population in northwestern Yunnan Province, China. We suspected that these intermediate individuals were the hybrids of R. humeana and R. cautleoides from the previous evidence, but could not confirm them. In this study, morphometric analysis was followed by examination of HAT-RAPD polymorphisms to determine the occurrence of natural hybridization between sympatric R. humeana and R. cautleoides. The results showed that most morphological characters of the putative hybrids were found to be intermediate between those of R. humeana and R. cautleoides. Meanwhile, molecular analysis confirmed that the morphological intermediates were derived from hybridization between the two species. From the analysis of the NewHybrids, the hybridization individuals were mainly F1s. These results indicated that interspecific hybridization between R. humeana and R. cautleoides indeed occurred in sympatric population.

Paraneoplastic neurological syndrome caused by cystitis glandularis: A case report and literature review.

BACKGROUND: Paraneoplastic neurological syndrome (PNS) is an unusual event. PNS caused by cystitis glandularis (CG) or a bladder tumor is extremely rare; hence, missed diagnosis or misdiagnosis can easily occur. To date, approximately 21 cases have been reported in PubMed. CASE SUMMARY: We report a case of PNS caused by CG and describe the clinical and imaging features. The main clinical feature was advanced cognitive impairment, and early clinical features were memory impairment, decreased computational ability, and abnormal behavior. Later clinical features were dementia, vomiting, inability to eat and walk, urinary incontinence, and hematuria. Imaging features on cranial magnetic resonance imaging were diffuse white matter lesions. Paraneoplastic tumor markers were normal. A total abdominal computed tomography scan showed multiple thickened areas on the bladder wall with local prominence. Cystoscopy revealed a volcanic protuberance on the posterior wall of the bladder with a diameter of 6 cm and no pedicle. The postoperative pathological diagnosis was CG. The patient recovered well following resection of CG. PNS cases caused by previous bladder tumors can be retrieved from PubMed to describe the clinical signs and prognosis of PNS. CONCLUSION: The main clinical feature of PNS caused by CG was dementia, and the imaging features were diffuse cerebral white matter lesions. Resection of CG lesions is the fundamental treatment for PNS induced by CG. This case highlights the importance of etiological treatment.

Functional implications of the staminal lever mechanism in Salvia cyclostegia (Lamiaceae).

BACKGROUND AND AIMS: Flower morphology and inflorescence architecture affect pollinator foraging behaviour and thereby influence the process of pollination and the reproductive success of plants. This study explored possible ecological functions of the lever-like stamens and the floral design in Salvia cyclostegia. METHODS: Flower construction was experimentally manipulated by removing either the lower lever arms or the upper fertile thecae of the two stamens from a flower. The two types of manipulated individuals were intermixed with the control ones and randomly distributed in the population. KEY RESULTS: Removing the sterile lower lever arms significantly reduced handling time per flower of the main pollinator, Bombus personatus. Interestingly, this manipulation did not increase the number of flowers probed per plant visit, but instead reduced it, i.e. shortened the visit sequence of the bumble-bees. Both loss of staminal lever function by removing lower lever arms and exclusion of self pollen by removing upper fertile thecae significantly reduced seed set per flower and seed set per plant. Both the manipulations interacted significantly with inflorescence size for the effect on female reproductive output. CONCLUSIONS: Though the intact flowers demand a long handling time for pollinators, the reversible staminal lever is of advantage by promoting dispersal of pollen and thus the male function. The particular floral design in S. cyclostegia contributes to the floral constancy of B. personatus bumble-bees, with the lower lever arms acting as an optical cue for foraging cognition.

Phylogeny and evolution of bracts and bracteoles in Tacca (Dioscoreaceae).

Most species in the genus Tacca (Dioscoreaceae) feature green to black purple, conspicuous inflorescence involucral bracts with variable shapes, motile filiform appendages (bracteoles), and diverse types of inflorescence morphology. To infer the evolution of these inflorescence traits, we reconstructed the molecular phylogeny of the genus, using DNA sequences from one nuclear, one mitochondrial, and three plastid loci (Internal Transcribed Spacer (ITS), atpA, rbcL, trnL-F, and trnH-psbA). Involucres and bracteoles characters were mapped onto the phylogeny to analyze the sequence of inflorescence trait evolution. In all analyses, species with showy involucres and bracteoles formed the most derived clade, while ancestral Tacca had small and plain involucres and short bracteoles, namely less conspicuous inflorescence structures. Two of the species with the most elaborate inflorescence morphologies (T. chantrieri in southeast China and T. integrifolia in Tibet), are predominantly self-pollinated, indicating that these conspicuous floral displays have other functions rather than pollinator attraction. We hypothesize that the motile bracteoles and involucres may facilitate selfing; display photosynthesis in the dim understory, and protect flowers from herbivory.

Chloroplast Genome Evolution in Four Montane Zingiberaceae Taxa in China.

Chloroplasts are critical to plant survival and adaptive evolution. The comparison of chloroplast genomes could provide insight into the adaptive evolution of closely related species. To identify potential adaptive evolution in the chloroplast genomes of four montane Zingiberaceae taxa (Cautleya, Roscoea, Rhynchanthus, and Pommereschea) that inhabit distinct habitats in the mountains of Yunnan, China, the nucleotide sequences of 13 complete chloroplast genomes, including five newly sequenced species, were characterized and compared. The five newly sequenced chloroplast genomes (162,878-163,831 bp) possessed typical quadripartite structures, which included a large single copy (LSC) region, a small single copy (SSC) region, and a pair of inverted repeat regions (IRa and IRb), and even though the structure was highly conserved among the 13 taxa, one of the rps19 genes was absent in Cautleya, possibly due to expansion of the LSC region. Positive selection of rpoA and ycf2 suggests that these montane species have experienced adaptive evolution to habitats with different sunlight intensities and that adaptation related to the chloroplast genome has played an important role in the evolution of Zingiberaceae taxa.

Sexual conflict in protandrous flowers and the evolution of gynodioecy.

Sexual interference between male and female function in hermaphrodite plants is reduced by protandry. In environments with insufficient pollinator service, prolongation of male function owing to limited pollen removal could restrict the duration of female function and lower seed production. We provide evidence that this form of sexual conflict has played a role in the spread of females in gynodioecious populations of Cyananthus delavayi in the pollen-limited environments in which this subalpine species occurs. Using field experiments involving artificial pollen removal from the strongly protandrous flowers of hermaphrodites, we demonstrated a trade-off between male- and female-phase duration with no influence on overall floral longevity. Pollen removal at the beginning of anthesis resulted in hermaphrodite seed production matching that of females. In contrast, restricted pollen removal increased the duration of male function at the expense of female function lowering maternal fertility compared to females. This pattern was evident in five populations with females experiencing a twofold average seed fertility advantage compared to hermaphrodites. Gynodioecy often appears to evolve from protandrous ancestors and pollen limitation is widespread in flowering plants suggesting that sexual conflict may play an unappreciated role in the evolution of this form of sexual dimorphism.

Nectar robbers pollinate flowers with sexual organs hidden within corollas in distylous Primula secundiflora (Primulaceae).

Nectar robbers are thought rarely to pollinate flowers, especially those with sexual organs hidden within corollas. In this study, we examined whether robbers pollinate flowers of distylous Primula secundiflora. Distylous plants have two floral morphs. Pin flowers have long styles and short stamens, and thrum flowers have short styles and long stamens. Flowers of P. secundiflora were commonly robbed by bumble-bees, and robbing holes were always situated between high and low sexual organs for both floral morphs. We observed that pollen grains of pin flowers were removed while thrum flowers received fresh pollen grains immediately after flowers were robbed. We manipulated flowers so that only nectar robbers could visit them. This resulted in 98 per cent of thrum flowers and 6 per cent of pin flowers setting fruit, and seed number per thrum fruit was also significantly higher than per pin fruit. Our findings suggest that nectar robbers transfer pollen from pin flowers to thrum flowers effectively, and consequently increase male fitness of the pin morph and female fitness of the thrum morph. Such asymmetrical pollen flow caused by nectar robbers may act as an important selective agent in floral fitness and evolution of distyly.

[A novel mutation of the PAX6 gene in a Chinese family with aniridia].

OBJECTIVE: The PAX6 gene encodes a transcriptional regulator involved in oculogenesis and other developmental processes such as aniridia, a congenital condition characterized by the underdevelopment of the iris of eyes. The function of the PAX6 gene in these two conditions is still poorly defined. The purpose of this study is to identify the mutation of the PAX6 gene in a Chinese family with aniridia. METHODS: Two aniridia patients collected from the family underwent full ophthalmologic examination. Genomic DNA was prepared from venous leukocytes of the two patients and five healthy individuals in the family, and 100 unrelated healthycontrols. Exons 4-13 and their immediate flanking sequences of the PAX6 gene was analyzed by PCR amplification, direct sequencing, and single-strand conformation polymorphism(SSCP). RESULTS: The sequencing result revealed a novel PAX6 mutation in the two patients. It was a heterozygous mutation (IVS10+1G>A) at the boundary of exon 10 and intron 10. The mutation was also detected by SSCP analysis. It was not detected in the healthy relatives and unrelated controls. CONCLUSION: Aniridia is an autosomal dominant inheritable disease. A novel PAX6 gene mutation has been identified in the Northeastern Chinese family with aniridia. The genetic analysis suggested that this novel mutation in the PAX6 gene is capable of causing the classic aniridia phenotype.