Fluorescein-labeled glutathione to study protein S-glutathionylation

Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins.     

The role of dyadic interactions in the mixed-mating strategies of the mangrove rivulus Kryptolebias marmoratus

The mangrove rivulus Kryptolebias marmoratus a small cyprinodont fish native to tropical and subtropical waters from Florida to Brazil, is one of two known self-fertilizing, hermaphroditic vertebrates of which K. marmoratus displays androdioecy, a complex system of reproduction in which hermaphrodites and males are present. This study describes the behavioral repertoires observed during dyadic interactions in the laboratory. Kryptolebias marmoratus exhibited 23 distinctive acts or behaviors. Acts were divided into four categories: aggressive, submissive, neutral, and reproductive. Leading and following behaviors played important roles in the behavioral repertoires of these fish. In hermaphrodite-male dyads, males exclusively initiated the reproductive process and actively pursued hermaphrodites. When hermaphrodites were paired, there was no evidence that they behaved like other simultaneous hermaphrodites that alternate sexual roles (e.g. serranids). Hermaphrodites were extremely aggressive towards one another, and the aggressor established dominance rapidly. Male-male dyads were divided into two subdivisions based on the presence or absence of the caudal ocellus on one fish. A caudal ocellus on one male appeared to signal the possibility of a potential mating partner to males lacking it. Pairings of males without an ocellus were similar to hermaphrodite-hermaphrodite dyads in that both members of the pair were aggressive towards one another. These observations may be indicative of interactions taking place in natural communities or assemblages of fish in which both males and hermaphrodites occur and provide evidence on the role of dyadic interactions in the mixed-mating strategies [Current Zoology 56 (1): 6-17, 2010].     

EGFR/Met association regulates EGFR TKI resistance in breast cancer

Breast cancers show a lack of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), despite 30% of tumors expressing EGFR. The mechanism of this resistance is unknown; however, we have recently shown that Met kinase activity compensates for loss of EGFR kinase activity in cell culture models. Met has been implicated in the pathogenesis of breast tumors and therefore may cooperate with EGFR for tumor growth. Here we have found that EGFR phosphorylation and cell proliferation is in part regulated by Met expression. In addition, we found that Met constitutive phosphorylation occurred independent of the Met ligand hepatocyte growth factor (HGF). Ligand-independent Met phosphorylation is mediated by Met amplification, mutation, or overexpression and by Met interaction with other cell surface molecules. In SUM229 breast cancer cells, we found that Met was not amplified or mutated, however it was overexpressed. Met overexpression did not directly correlate with ligand-independent Met phosphorylation as the SUM229 cell line was the only Met expressing breast cancer line with constitutive Met phosphorylation. Interestingly, Met expression did correlate with EGFR expression and we identified an EGFR/Met complex via co-immunoprecipitation. However, we only observed Met constitutive phosphorylation when c-Src also was part of this complex. Ligand-independent phosphorylation of Met was decreased by down regulating EGFR expression or by inhibiting c-Src kinase activity. Lastly, inhibiting EGFR and Met kinase activities resulted in a synergistic decrease in cell proliferation, supporting the idea that EGFR and Met functionally, as well as physically interact in breast cancer cells to regulate response to EGFR inhibitors.     

Distinguishing cell types or populations based on the computational analysis of their infrared spectra

Infrared (IR) spectroscopy of intact cells results in a fingerprint of their biochemistry in the form of an IR spectrum; this has given rise to the new field of biospectroscopy. This protocol describes sample preparation (a tissue section or cytology specimen), the application of IR spectroscopy tools, and computational analysis. Experimental considerations include optimization of specimen preparation, objective acquisition of a sufficient number of spectra, linking of the derived spectra with tissue architecture or cell type, and computational analysis. The preparation of multiple specimens (up to 50) takes 8 h; the interrogation of a tissue section can take up to 6 h (～100 spectra); and cytology analysis (n = 50, 10 spectra per specimen) takes 14 h. IR spectroscopy generates complex data sets and analyses are best when initially based on a multivariate approach (principal component analysis with or without linear discriminant analysis). This results in the identification of class clustering as well as class-specific chemical entities.     

Introduction of customized inserts for streamlined assembly and optimization of BioBrick synthetic genetic circuits

Background  

BioBrick standard biological parts are designed to make biological systems easier to engineer (e.g. assemble, manipulate, and modify). There are over 5,000 parts available in the Registry of Standard Biological Parts that can be easily assembled into genetic circuits using a standard assembly technique. The standardization of the assembly technique has allowed for wide distribution to a large number of users -- the parts are reusable and interchangeable during the assembly process. The standard assembly process, however, has some limitations. In particular it does not allow for modification of already assembled biological circuits, addition of protein tags to pre-existing BioBrick parts, or addition of non-BioBrick parts to assemblies.     

Development of a molecular diagnostic system to discriminate Dreissena polymorpha (zebra mussel) and Dreissena bugensis (quagga mussel)

A 3-primer PCR system was developed to discriminate invasive zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel. The system is based on: 1) universal primers that amplifies a region of the nuclear 28s rDNA gene from both species and 2) a species-specific primer complementary to either zebra or quagga mussel. The species-specific primers bind to sequences between the binding sites for the universal primers resulting in the amplification of two products from the target species and one product from the nontarget species. Therefore, nontarget products are positive amplification controls. The 3-primer system accurately discriminated zebra and quagga mussels from seven geographically distinct populations.     

Prostaglandin-E2 is produced by adult human epidermal melanocytes in response to UVB in a melanogenesis-independent manner

Excessive ultraviolet radiation (UVR) exposure induces erythema, mediated in part by prostaglandin-E₂ (PGE₂). While keratinocytes are a major PGE₂ source, epidermal melanocytes (EM) also express PGE₂-production machinery. It is unclear whether EM-produced PGE₂ contributes to UVR-induced skin inflammation, and whether this is correlated with melanogenesis. Epidermal melanocytes were cultured from skin phototype-1 and -4 donors, followed by assessment of PGE₂ production and melanogenesis. Epidermal melanocytes expressed cytoplasmic phospholipase-A₂, cyclooxygenase-1, cytoplasmic prostaglandin-E synthase and microsomal prostaglandin-E synthase-1, -2. Epidermal melanocytes produced PGE₂ under basal conditions, which increased further after arachidonic acid stimulation. Epidermal melanocytes expressed cyclooxygenase-2 (COX-2) mRNA and a selective COX-2 inhibitor (NS-398) reduced PGE₂ production. Ultraviolet B-induced PGE₂ production was positively correlated with skin phototype-1, despite variability between individual EM donors. By contrast, there was no correlation between PGE₂ production by EM and their melanogenic status. Thus, EM may contribute to UVR-induced erythema, with role of donor skin phototype more important than their melanogenic status.     

Effects of pup age,maternal age,and birth date on pre‐weaning survival rates of Weddell seals in Erebus Bay,Antarctica

Identifying factors affecting juvenile survival is important to understanding the dynamics of populations and may also provide insights into life history theory and the selective forces shaping evolution. Quantifying the relative influence of the various potential selective forces for the post‐birth, maternal dependency, and independent periods is difficult and often limits investigators to estimating a single juvenile survival rate for the first year of life, or from birth to recruitment. We examined survival of individually marked Weddell seal Leptonychotes weddellii pups during the maternal dependency period in Erebus Bay, Antarctica from 2005–2007. We used mark‐recapture models to evaluate competing a priori hypotheses regarding variation in daily pre‐weaning survival rates (φ) during the first 42 days of age. The a priori model receiving the most support from the data supported several of our predictions: increased with pup age and was higher for pups born later in the season and to older mothers. Increases in with increasing pup age may have been due to improved resilience to the environment, phenotypic selection against the frailest pups, or both. Maternal age was more important to than was maternal experience or age of primiparity, potentially indicating that age‐related increases in body mass allow greater offspring provisioning, or age‐related improvements in competitive abilities benefit offspring during the period of maternal care. Depending on the timing of birth and the age of the mother, ^{42 days} ranged from 0.79 (SE = 0.05) to 0.98 (SE = 0.01). These exceptionally high pre‐weaning survival rates contrast with estimates from other large terrestrial and marine mammal species where neonate survival is considerably lower and suggest that in species with similar life histories, pre‐weaning survival probability may be buffered from both predators and environmental fluctuations during the period of maternal nutritional dependency. Climatic changes affecting stability of ice used for pupping substrate or extent of fast‐ice buffering pupping colonies from predators have the potential to reduce pre‐weaning survival and may have important implications for population growth rates.     

Soil carbon and nitrogen mineralization following deposition of insect frass and greenfall from forests under elevated CO2 and O3

Elevated CO₂ and O₃ alter tree quality and the quality of herbivore inputs, such as frass, to forest soil. Altered quality or quantity of herbivore inputs to the forest floor can have large impacts on belowground processes. We collected green leaves and frass from whitemarked tussock moth caterpillars from aspen-birch stands at the Aspen Free Air CO₂ Enrichment (FACE) site near Rhinelander, WI, USA. Small or large quantities of frass, greenfall, or a 1:1 ratio of frass and greenfall were added to microcosms for each FACE treatment (control, +CO₂, +O₃, +CO₂+O₃). We measured initial frass and greenfall quality, and recorded microbial respiration, and nitrate leaching over 40 days. Elevated carbon dioxide (eCO₂) and tropospheric ozone (eO₃) significantly altered the carbon, nitrogen, and condensed tannin content of insect frass and green leaves. Although FACE treatments affected input quality, they had minimal effect on microbial respiration and no effect on nitrogen leaching. In contrast, input quantity substantially influenced microbial respiration and nitrate leaching. Respiratory carbon loss and nitrate immobilization were nearly double in microcosms receiving large amounts of herbivore inputs than those receiving no herbivore inputs. Small amounts of herbivore inputs, however, did not significantly alter microbial respiration or immobilization, suggesting that effects of herbivore inputs on soil processes will be detected only at moderate to high herbivory/input levels. These results suggest that subtle changes in frass and greenfall quality may not affect soil nutrient cycling. In contrast, environmental change induced increases in insect population size or frass and greenfall inputs to the soil may substantially impact nutrient cycling.