Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

Receptor inactivation by dye-neuropeptide conjugates. 3. Comparative binding of dye-neuropeptide conjugates to FMRFamide receptors of Helix aspersa and Loligo pealei

Three neuropeptide analogues of FMRFamide (FMRFa) were covalently attached to a tethered derivative of methylene blue to form dye-neuropeptide conjugates. The comparative binding of the latter to FMRFa receptors was subsequently examined in both Helix aspersa (circumesophageal ganglia) and squid (optic lobe membrane). In Helix, the FMRFa analogue CFMRFamide (CFMRFa) inhibited the specific binding of the FMRFa ligand [¹²⁵I]daYFnLRFa in a dose-dependent manner. Az-CFMRFa, one of the dye-neuropeptide conjugates, also dose-dependently inhibited the specific binding of [¹²⁵I]daYFnLRFa. Moreover, their potencies equaled or exceeded that of FMRFamide. In squid, the binding of CFMRFa and FMRFa was similar. However, the dye-neuropeptide conjugate (IC₅₀ of 14 nM) was about 44-fold less potent than FMRFa. The conjugates were synthesized as part of a study seeking to target and inactivate preselected receptors with heretofore unattainable selectivity and permanence.     

A lack of locomotor activity rhythms inDrosophila melanogaster larvae (Diptera: Drosophilidae)

We examined the locomotor activity ofDrosophila melanogaster for the existence of circadian rhythms, using the wild type and two mutants of theperiod (per) gene,per ^o andper ^s. This was accomplished using a newly described apparatus for the recording and measurement of larval path lengths over a 96-h test period. None of the larvae examined exhibited appreciable diel rhythms under cycling conditions of light or temperature. Larvae were also not rhythmic under free-running conditions. Our results suggest that theper gene does not influence an observable locomotor behavioral phenotype in the larval stage of development.     

Cardiovascular and metabolic responses to noradrenaline in men acclimatized to cold baths

The purpose of this study was to see whether artificial acclimatization to cold would reduce the pressor response to noradrenaline (NA) as natural acclimatization has been shown to do, and whether it would induce nonshivering thermogenesis. Three white men were infused with NA at four dosage levels between 0.038 and 0.300 g·kg^–1·min^–1 (2–23 g·min^–1), before and after artificial acclimatization to cold and again 4 months later when acclimatization had decayed. Acclimatization was induced by ten daily cold (15°Q baths of 30–60 min followed by rapid rewarming in hot (38–42°C) water, and was confirmed by tests of the subjects responses to whole-body cooling in air. Three control subjects also underwent the first and third tests. Acclimatization substantially reduced the pressor response to NA at 0.150 and 0.300 g·kg^–1·min^–1, confirming earlier findings by the same technique in naturally acclimatized men, and its decay increased this response to beyond its initial levels (P<0.05 for both changes). Acclimatization did not change the response to NA of heart rate, subjective impressions, skin temperature of finger and toe, pulmonary ventilation, or plasma free fatty acids and ketone bodies. At no time did NA increase oxygen consumption, or increase skin temperature or heat flow over reported sites of brown fat. These findings would seem to show that acclimatization to cold reduces sensitivity to the pressor effect of NA but does not induce nonshivering thermogenesis, and that the reduced sensitivity is replaced by a hypersensitivity to NA when acclimatization decays.     

Change in climatically suitable breeding distributions reduces hybridization potential between Vermivora warblers

Aim

Climate change is affecting the distribution of species and subsequent biotic interactions, including hybridization potential. The imperiled Golden-winged Warbler (GWWA) competes and hybridizes with the Blue-winged Warbler (BWWA), which may threaten the persistence of GWWA due to introgression. We examined how climate change is likely to alter the breeding distributions and potential for hybridization between GWWA and BWWA.

Location

North America.

Methods

We used GWWA and BWWA occurrence data to model climatically suitable conditions under historical and future climate scenarios. Models were parameterized with 13 bioclimatic variables and 3 topographic variables. Using ensemble modeling, we estimated historical and modern distributions, as well as a projected distribution under six future climate scenarios. We quantified breeding distribution area, the position of and amount of overlap between GWWA and BWWA distributions under each climate scenario. We summarized the top explanatory variables in our model to predict environmental parameters of the distributions under future climate scenarios relative to historical climate.

Results

GWWA and BWWA distributions are projected to substantially change under future climate scenarios. GWWA are projected to undergo the greatest change; the area of climatically suitable breeding season conditions is expected to shift north to northwest; and range contraction is predicted in five out of six future climate scenarios. Climatically suitable conditions for BWWA decreased in four of the six future climate scenarios, while the distribution is projected to shift east. A reduction in overlapping distributions for GWWA and BWWA is projected under all six future climate scenarios.

Main Conclusions

Climate change is expected to substantially alter the area of climatically suitable conditions for GWWA and BWWA, with the southern portion of the current breeding ranges likely to become climatically unsuitable. However, interactions between BWWA and GWWA are expected to decline with the decrease in overlapping habitat, which may reduce the risk of genetic introgression.     

Intratumoral interleukin-2 immunotherapy: Activation of tumor-infiltrating and splenic lymphocytes in vivo

Direct intratumoral injection of interleukin-2 (IL-2) was evaluated in a murine model. Balb/c mice received 5 × 10⁴ Line 1 alveolar carcinoma cells (L1C2) by subcutaneous injection. On the third day following tumor implantation, mice received injections of IL-2 (5 × 10³–5 × 10⁴ units) or diluent twice daily, either by i. p. or intratumoral injection, 5 days/week for 3 weeks. Intratumoral injection of 5 × 10⁴ units IL-2 significantly reduced tumor volume (P <0.05 versus control), increased median survival time (P = 0.0001), and resulted in a 23.5% cure rate (P = 0.008). There were no long-term survivors in the other treatment groups. Both tumor-infiltrating lymphocytes (TIL) and splenic lymphocytes isolated directly from IL-2-treated mice demonstrated enhanced cytolytic activity compared to diluent-treated controls. To determine whether non-T-cell-mediated antitumor responses were active in our model, intratumoral immunotherapy was evaluated in athymic Balb/cnu/nu mice. In order to decrease the recruitment of lymphocyte precursors, nude mice were splenectomized and received cyclophosphamide prior to tumor injection and IL-2 therapy. Intratumoral IL-2 immunotherapy also significantly decreased tumor volume in these immunodeficient mice (P <0.02), but did not lead to long-term survival. We conclude that both TIL and splenic lymphocytes are activated in vivo in response to intratumoral IL-2 immunotherapy, suggesting that intratumoral therapy with IL-2 activates both local and systemic antitumor responses.Supported by the Tobacco-Related Disease Research Program of the University of California, the Cancer Research Coordinating Committee, the Jonsson Cancer Center Foundation, and Veterans Administration Medical Research Funds     

Use of 31P magnetisation transfer magnetic resonance spectroscopy to measure ATP changes after 670 nm transcranial photobiomodulation in older adults

Mitochondrial function declines with age, and many pathological processes in neurodegenerative diseases stem from this dysfunction when mitochondria fail to produce the necessary energy required. Photobiomodulation (PBM), long-wavelength light therapy, has been shown to rescue mitochondrial function in animal models and improve human health, but clinical uptake is limited due to uncertainty around efficacy and the mechanisms responsible. Using ³¹P magnetisation transfer magnetic resonance spectroscopy (MT-MRS) we quantify, for the first time, the effects of 670 nm PBM treatment on healthy ageing human brains. We find a significant increase in the rate of ATP synthase flux in the brain after PBM in a cohort of older adults. Our study provides initial evidence of PBM therapeutic efficacy for improving mitochondrial function and restoring ATP flux with age, but recognises that wider studies are now required to confirm any resultant cognitive benefits.     

Novel thieno[2,3-b]- and [3,4-b]pyrans as potassium channel openers. Thiophene systems—XVII

The syntheses and antihypertensive activity of the thieno[3,4-b]pyran and thieno[2,3-b]pyran isosteres of the potassium channel opener (PCO) RWJ 26629 (± 2a) are reported. While the unsubstituted thiophene derivatives were active at 20 mg/kg, introduction of a strong electron withdrawing group in the 2-position of the thieno[3,2-b] series increased potency. Similar substitution on the thieno[3,4-b] series significantly lowered potency. Compounds 26 and 30 are approximately 5-fold more potent than the prototypic PCO cromakalim (± 1).