Casein Accumulation by Rat Mammary Epithelial Cells Grown within a Reconstituted Basement Membrane Is Modulated by Fatty Acids in a Hormone- and Time-Dependent Manner

The mammary gland is under complex regulation involving the participation of hormones, growth factors, and stromal components, including lipids. Our laboratory has developed a unique primary culture system that allows undifferentiated mammary epithelial cells from immature virgin rats to proliferate and differentiate to an extent equivalent to the lactating mammary gland. Using this model system we have examined the effects of the unsaturated fatty acids oleate and linoleate on mammary epithelial cell proliferation as well as both morphological and functional differentiation. Neither fatty acid showed any effect on cell proliferation whether added to cells in the presence of optimal serum-free medium or under suboptimal conditions of epidermal growth factor (EGF) and prolactin. Morphological differentiation also was not affected by fatty acid addition under either optimal or suboptimal conditions, although a decrease was observed when medium depleted in EGF and prolactin was compared to optimal medium. The notable finding in this study was that both oleate and linoleate modulated functional differentiation, as assessed by casein accumulation, in a time- and hormone-dependent manner. At early times in culture, casein levels were stimulated by both oleate and linoleate; this effect was most dramatic under suboptimal conditions of prolactin and EGF. In marked contrast, however, linoleate decreased casein levels by approximately 50% in optimal medium, at all concentrations tested, after at least 7 days in culture. This decrease was also observed in suboptimal medium, although the concentration of EGF and prolactin influenced the extent of the reduction. Although the mechanism is currently unknown, it is tempting to speculate that the cellular and biochemical events that result in linoleate-induced inhibition of functional differentiation may also be involved in the tumor-enhancing properties of this fatty acid.     

Site specific labelling of sugar residues in oligoribonucleotides: reactions of aliphatic isocyanates with 2' amino groups.

Considerable effort has been directed towards studying the structure and function of nucleic acids and several approaches rely on the attachment of reporter groups or reactive functional groups to nucleic acids. We report here the selective modification of 2-amino groups in oligoribonucleotides, through their reaction with aliphatic isocyanates, to give the corresponding 2'-urea derivatives in >95% yield. Furthermore, such modification with (2-isocyanato)ethyl 2-pyridyl disulfide enables subsequent coupling to other thiols (such as those contained in peptides and proteins) or to thiol-reactive electrophiles. A modified decamer was not significantly destabilized by the 2'-urea group, compared with a 2'-amino group, as demonstrated by a mere 1.3 degrees C drop in the melting temperature of the duplex.     

Species status of Neisseria gonorrhoeae: evolutionary and epidemiological inferences from multilocus sequence typing

Background  

Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species.     

Induction of interleukin-1/and interleukin-8 mRNAs and proteins by TGFβ1 in rat lung alveolar epithelial cells

The transforming growth factor-β (TGF-β) has been shown to increase in lung injury and in fibrotic states of the lung. In the current study, we sought to investigate whether TGFβ₁ induced the expression of IL-1α and IL-8 in rat alveolar epithelial cells. We evaluated TGFβ₁, IL-1α, and IL-8 expression by immunofluorescence in silica-injured and saline-treated control rat lungs. Antibodies to IL-1α, IL-8, and TGFβ₁ showed intense staining in silica-injured lungs as compared to saline-instilled lungs. Primary isolated type II cells from silica-injured lungs showed increased expression of IL-1α as compared to saline-instilled lungs. To evaluate the effects of TGFβ₁, we treated an immortalized rat type II cell-derived cell line (LM5) with 100 pg/ml of TGFβ₁ in serum-free medium for 0–24 hours and analyzed the expression of IL-1α and IL-8 mRNAs and proteins using semi-quantitative RT-PCR, Northern blot analysis, Western blot analysis, and immunohistochemistry. Densitometric analysis of Northern blots showed modest constitutive expression of IL-1α gene in untreated control LM5 cells. TGFβ₁ treatment resulted in an increase in IL-1α mRNA, that reached maximum levels (4-fold) by 2 hours and remained elevated for 4–16 hours, with a subsequent decline by 24 hours. Similarly, Northern blot and RT-PCR analysis demonstrated that TGFβ₁ treatment resulted in maximum induction of IL-8 mRNA (6–8.5-fold) within 1–4 hours. The levels remained elevated for up to 24 hours afterwards. Western blot analysis results further confirmed the expression of both IL-1α and IL-8 proteins by LM5 cells. TGFβ₁ treatment resulted in increased expression of both IL-1α and IL-8 proteins. Immunofluorescence studies demonstrated increased staining of IL-1α by TGFβ₁ as compared to untreated cells. These results suggest that TGFβ₁ may regulate IL-1α and IL-8 expression in alveolar epithelial cells and contribute to polymorphonuclear leukocyte recruitment and lung injury in clinical states with increased TGFβ₁. © 1996 Wiley-Liss, Inc.     

Distinct conformers of amyloid beta accumulate in the neocortex of patients with rapidly progressive Alzheimer's disease

Amyloid beta (Aβ) deposition in the neocortex is a major hallmark of Alzheimer''s disease (AD), but the extent of deposition does not readily explain phenotypic diversity and rate of disease progression. The prion strain–like model of disease heterogeneity suggests the existence of different conformers of Aβ. We explored this paradigm using conformation-dependent immunoassay (CDI) for Aβ and conformation-sensitive luminescent conjugated oligothiophenes (LCOs) in AD cases with variable progression rates. Mapping the Aβ conformations in the frontal, occipital, and temporal regions in 20 AD patients with CDI revealed extensive interindividual and anatomical diversity in the structural organization of Aβ with the most significant differences in the temporal cortex of rapidly progressive AD. The fluorescence emission spectra collected in situ from Aβ plaques in the same regions demonstrated considerable diversity of spectral characteristics of two LCOs—quatroformylthiophene acetic acid and heptaformylthiophene acetic acid. Heptaformylthiophene acetic acid detected a wider range of Aβ deposits, and both LCOs revealed distinct spectral attributes of diffuse and cored plaques in the temporal cortex of rapidly and slowly progressive AD and less frequent and discernible differences in the frontal and occipital cortex. These and CDI findings indicate a major conformational diversity of Aβ accumulating in the neocortex, with the most notable differences in temporal cortex of cases with shorter disease duration, and implicate distinct Aβ conformers (strains) in the rapid progression of AD.     

Quirks of dye nomenclature. 8. Methylene blue,azure and violet

Methylene blue was synthesized in 1877 and soon found application in medicine, staining for microscopy and as an industrial dye and pigment. An enormous literature has accumulated since its introduction. Early on, it was known that methylene blue could be degraded easily by demethylation; consequently, the purity of commercial samples often was low. Therefore, demethylation products, such as azures and methylene violet, also are considered here. The names and identity of the components, their varying modes of manufacture, analytical methods and their contribution to biological staining are discussed.     

Selective breeding of entomopathogenic nematodes for enhanced attraction to a root signal did not reduce their establishment or persistence after field release

We recently showed that the efficacy of an entomopathogenic nematode (EPN) as a biological control agent against a root pest could be enhanced through artificial selection. The EPN Heterorhabditis bacteriophora was selected for higher responsiveness towards (E)-β-caryophyllene (EβC), a sesquiterpene that is emitted by maize roots in response to feeding damage by the western corn rootworm (WCR). EβC is normally only weakly attractive to H. bacteriophora, which is one of the most infectious nematodes against WCR. By selecting H. bacteriophora to move more readily along a EβC gradient we obtained a strain that was almost twice more efficient in controlling WCR population in fields planted with an EβC-producing maize variety. However, artificial selection for one trait may come at a cost for other important traits such as infectiousness, establishment and/or persistence in the field. Indeed, infectiousness was slightly but significantly reduced in the selected strain. Yet, this apparent cost was largely compensated for by the higher responsiveness to the root signal. Here we show that the selection process had no negative effect on establishment and persistence of field-released EPN. This knowledge, combined with the previously reported results, attest to the feasibility of manipulating key traits to improve the efficacy of beneficial organisms.Key words: entomopathogenic nematodes, tritrophic interactions, artificial selection, biological control, Diabrotica virgifera virgifera, western corn rootworm, persistence, establishmentDiabrotica virgifera virgifera LeConte (Chrysomelidae: Coleptera, western corn rootworm, WCR) is a major well established pest of maize in the American Corn Belt and more recently also in Europe.¹ The larval stages of this beetle can cause significant damages to maize roots, leading to reduction of plant growth, deficiencies in nutrient and water uptake, lodging, increased susceptibility to water stress and reduced grain yield.² This combination of factors result in an estimated loss of one billion US dollars per year in the USA.³ The pest has been introduced in Europe in the early ''90s,⁴ and it is expected that at full establishment the costs resulting from WCR damages will be half a billion Euros.⁵ Several strategies are available to control this soil-dwelling pest, including crop rotation, pesticides and transgenic Bt maize, but WCR can readily evolve resistance to each of these methods.^6–8 This is why efforts have been invested in biological control alternatives.Entomopathogenic nematodes (EPN) show great promise as biological agents against WCR.⁹ Root-produced volatiles appear to play an important role in the recruitment of EPN^10–13 and one such volatile, (E)-β-caryophyllene (EβC), has recently been identified for maize roots¹⁴ and was found to be an ideal below-ground alarm signal.¹⁵ EPN efficacy can be improved by exploiting the ability of WCR-damaged maize roots to emit the attractant.¹⁴ Further studies have shown the importance of choosing the right species of nematodes.¹⁶ Among the EPN species tested against WCR, Heterorhabditis bacteriophora has proven to be one of the most virulent nematodes,¹⁷ but it barely responds to EβC.¹⁶ We therefore recently selected H. bacteriophora for higher responsiveness to EβC.¹⁸ In the field, the selected strain exhibited better abilities to control WCR larvae, but logically only in maize plots with plants that emitted EβC. However, previous studies have shown that enhancing beneficial traits through selective breeding can incur costs and negatively alter other traits in the selected strain.¹⁹ For EPN such trade-offs after selective breeding have also been reported, for instance resulting in reduced storage stability²⁰ or a lower capacity to kill their hosts.²¹ After selection for enhanced responsiveness to EβC response, we observed a small, but significant negative effect on infectiousness of the selected strains. However, this drawback was readily outweighed by the improved ability to locate hosts in the field.¹⁸Not only infectiousness is a crucial trait for the successful use of EPN in biological control: establishment and persistence in the field are of decisive importance as well. These traits vary with EPN species and are determined by biotic factors such as pathogens and predators²² or abiotic factors such as soil type,²³ humidity,²⁴ temperature²⁵ or pH.²⁴ But the main factor that is thought to determine long-term persistence in the field is the presence of available host insects.²⁵ In field trials in Hungary, three EPN species, H. bacteriophora, H. megidis and Steinernema feltiae, were released to test their control potential against WCR. They all persisted at least as long WCR were present in soil, during the same year.²⁶ There was no significant difference between the three species in the establishment or persistence. Yet, independent of timing of application, EPN populations dramatically decreased within five months after application. The authors²⁶ propose that this short persistence is due to the absence of suitable alternative hosts in intensively cultivated crop fields in Europe.To determine if the selection for enhanced responsiveness to EβC went at a cost for establishment and persistence we compared these key traits for the original and the EβC-selected stains. Using a metal auger (2 cm diam.; 20 cm high), 310 soil samples were dug out either two days (establishment) or 28 days (persistence) after EPN application. The soil was placed in plastic boxes (4.5 cm diam.; 60 cm high) and as previously described²⁶ Tenebrio molitor (Coleoptera: Tenebrionidae) larva was placed as bait in the boxes. Presence/absence of EPN was evaluated by visually checking T. molitor larvae for EPN infection. Soil samples from areas where no EPN were applied served as controls. No significant differences were found between the original and selected strain of H. bacteriophora strain (factor “strain”), neither in establishment after two days nor in persistence after 28 days (factor “time”) (Fig. 1, two-way ANOVA, Ftime_1,35 = 2.937, p = 0.097; Fstrain_2,35 = 10.359, p < 0.001; Ftime × strain_2,35 = 1.202, p = 0.315, statistical differences within factors were calculated using a Bonferoni post-hoc test). Hence, the selection of H. bacteriophora for a better response to EβC had no consequence for how the nematodes settled in the experimental fields. Future efforts to improve the effectiveness H. bacteriophora against WCR might also include selection for increased persistence in soil. This would allow lower application rates and could provide growers with an affordable and efficient control strategy against this voracious pest.Open in a separate windowFigure 1Establishment and persistence of the original and a selected strain of H. bacteriophora. The selected strain (squares) established and persisted as well as the original strain (diamonds). The triangles represent control samples from plots where no nematodes were released. Establishment (after two days) and persistence (after 28 days) was equal for both strains. Moreover, the number of soil samples containing EPN after 28 days was not significantly lower than after 2 days, independently of treatment. A few nematodes were detected in the control samples but again no differences over time were detected. Error bars indicate the SEM. Different lower-case letters indicate statistical differences within establishment (after 2 days) or persistence (after 28 days) (p <0.05).So far, manipulation of tritrophic systems in order to improve biological control has been largely theoretical.^27–29 We show here that for EPN this approach is realistic and that their responsiveness to root-produced foraging signals can be enhanced without significant costs for other relevant traits. It has also been shown that the emissions of the signals by the plants can be enhanced.³⁰ Combining these strategies opens new perspectives for the development of ecologically sound strategies in pest management.     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.