Richard F. Kopp

Cutting cycle and spacing effects on biomass production by a willow clone in New York.

An experiment was established in central New York State in 1990 to determine cutting cycle and spacing effects on willow biomass production. Cutting cycles were annual, biennial and triennial, and spacings were 0.3 × 0.3, 0.3 × 0.9, and 0.6 × 1.1 m; biomass production and survival by willow clone SV1 (Salix dasyclados) were measured. Beginning in the second growing season, trees were fertilized with N, P and K, and irrigated. Willow clone SV1 harvested triennially with 0.3 × 0.9 m spacing yielded 71.3 odt ha−1, an average annual production of 23.8 odt ha−1 year−1. Spacing of 0.3 × 0.9 m yielded the most biomass, but spacing differences were not significant for biomass production. Triennial harvesting was significantly more productive than cumulative production after 3 years with annual harvesting (64.5 versus 39.2 odt ha−1). Cumulative production from two biennial harvests was significantly larger than cumulative production from four annual harvests (64.3 versus 50.1 odt ha−1). Tree survival was similar among cutting cycles after five growing seasons, averaging 75%. Statistically significant differences in survival were detected among spacings, averaging 88, 80 and 57% at 0.60 by 1.1, 0.3 × 0.9 and 0.3 × 0.3 m spacings, respectively during 1994.

Insulin Secretion and Ca2+ Dynamics in β-Cells Are Regulated by PERK (EIF2AK3) in Concert with Calcineurin

Background: A genetic deficiency in protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) in human and mice results in insulin-dependent diabetes. Results: Acute inhibition of PERK impairs insulin secretion, secretagogue-stimulated Ca2+ influx, and sarcoplasmic endoplasmic reticulum Ca2+-ATPase activity through a calcineurin-dependent pathway. Conclusion: PERK and calcineurin regulates Ca2+ dynamics underlying insulin secretion. Significance: Our findings provide insights into the intracellular mechanisms underlying stimulated insulin secretion. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting β-cell. Although genetic ablation of PERK in β-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent β-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca2+ signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca2+ entry and sarcoplasmic endoplasmic reticulum Ca2+-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates β-cell Ca2+ signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain.

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.

Predicting within-family variability in juvenile height growth of Salix based upon similarity among parental AFLP fingerprints

Abstract.Willow is being developed as a crop for biomass plantations in the Northeast and North-central United States, but has only recently been the subject of controlled breeding to generate improved genotypes. Maximizing variability among progeny within full-sib families produced by controlled pollination may increase the probability of producing willow clones exhibiting desirable extreme phenotypes. Yet, predicting combinations of parents yielding highly variable progeny is not currently possible. Controlled pollinations were completed among 15 Salix eriocephala clones and the resulting progeny were vegetatively propagated and planted in a greenhouse progeny test. Heights of rooted cuttings were measured after 4 months of growth. Genetic similarity among parents was estimated based on 77 polymorphic AFLP bands. Strong negative correlation (r = –0.88) was detected between mean female-parent similarity indices and the standard deviation of height among half-sib progeny from those females. Parent combinations that had relatively low similarity indices tended to produce progeny that had greater variability in height. This negative relationship suggests that AFLP fingerprints of S. eriocephala parents may be useful for predicting parent combinations that will yield families with large variability.

Collection and storage of pollen from Salix (Salicaceae).

Genetic improvement of willows through traditional breeding can be facilitated by pollen collection and storage so that female flower receptivity need not be synchronized with pollen shed for breeding. Two experiments were completed to test the effectiveness of various organic solvents for willow pollen collection. In the first experiment, seven pollen collection treatments and an untreated control were tested with two willow clones. The other experiment tested three treatments that showed promise in the initial experiment and an untreated control with eight willow clones. Toluene and carbon tetrachloride were effective for pollen extraction, with average pollen germination percentages that were >15%, but both chemicals reduced pollen viability by 10-20% compared with an untreated control based on in vitro germination tests. Pollen extracted with carbon tetrachloride or toluene was successfully used in controlled pollination, and >100 new families were produced with this technique. Pollen viability remained high after 18 mo of storage at -20°C. Based on our results, toluene is the preferred solvent for future willow pollen extractions because it is as effective as carbon tetrachloride, is not a known carcinogen, and is less expensive.

Quantitative Genetics of Traits Indicative of Biomass Production and Heterosis in 34 Full-sib F1Salix eriocephala Families

This project examined the heritability of traits that affect biomass production of Salix eriocephala, a shrub willow native to North America and an essential species in the breeding of bioenergy crop varieties. Using an incomplete factorial design, seven females and eight males were crossed to produce 34 full-sib F1 families. Five to 12 entries per family were planted in four-plant plots in a randomized complete block design on two sites. Melampsora rust incidence was scored in the fall of the first growing season (prior to coppice). Height of the tallest stem, cross-sectional stem area per stool, and number of stems per stool were recorded in the winter after the first growing season post-coppice. Plants were harvested 3xa0years post-coppice and biomass yield was determined. A large percentage of the total variance was additive for all of the traits studied and heritability estimates were low to moderate, suggesting that phenotypic expression of these traits is predictable and can be improved through breeding. Based on yield 3xa0years after coppice, 29 of the 34 families displayed midparent heterosis, ranging from 1–115%, for the composite trait—biomass yield, strongly indicating that offspring often perform better than their parents in this population. This study will assist in selecting parents which may produce superior progeny in the breeding program.

Resveratrol Interferes with Fura-2 Intracellular Calcium Measurements

Resveratrol, a naturally occurring polyphenol found in some fruits and especially in grapes, has been reported to provide diverse health benefits. Resveratrol’s mechanism of action is the subject of many investigations, and some studies using the ratiometric calcium indicator Fura-2 suggest that it modulates cellular calcium responses. In the current study, contradictory cellular calcium responses to resveratrol applied at concentrations exceeding 10xa0μM were observed during in vitro imaging studies depending on the calcium indicator used, with Fura-2 indicating an increase in intracellular calcium while Fluo-4 and the calcium biosensor YC3.60 indicated no response. When cells loaded with Fura-2 were treated with 100xa0μM resveratrol, excitation at 340xa0nm resulted in a large intensity increase at 510xa0nm, but the expected concurrent decline with 380xa0nm excitation was not observed. Pre-treatment of cells with the calcium chelator BAPTA-AM did not prevent a rise in the 340/380 ratio when resveratrol was present, but it did prevent an increase in 340/380 when ATP was applied, suggesting that the resveratrol response was an artifact. Cautious data interpretation is recommended from imaging experiments using Fura-2 concurrently with resveratrol in calcium imaging experiments.

Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) is Regulated by Store-Operated Ca2+ Entry and Mediates Cytoprotection Against Necrotic Cell Death

Background: Mechanisms underlying SGK1 activation are incompletely understood in epithelial cells. Results: Store-operated Ca2+ entry up-regulates SGK1, thereby modulating the lethal effects of Ca2+ overloading on mitochondrial membrane potential. Conclusion: Ca2+-induced SGK1 activates cytoprotective signaling and modifies mitochondrial function in epithelial cells. Significance: This work reveals a cytoprotective role for SGK1 in necrosis and has potential relevance for epithelial cell protection and cancer treatment. Serum and glucocorticoid-regulated kinase 1 (SGK1) encodes a phosphatidylinositol 3-kinase-dependent serine/threonine kinase that is rapidly induced in response to cellular stressors and is an important cell survival signal. Previous studies have suggested that an increase in cytoplasmic Ca2+ concentration ([Ca2+]c) is required for increased SGK1 expression, but the subcellular source of Ca2+ regulating SGK1 transcription remains uncertain. Activation of endoplasmic reticulum stress (ERS) with thapsigargin (TG) increased SGK1 mRNA and protein expression in MDA-MB-231 cells. Intracellular Ca2+ imaging revealed that store-operated Ca2+ entry played a prominent role in SGK1 induction by TG. Neither ERS nor release of Ca2+ from the ER was sufficient to activate SGK1. Prolonged elevation of intracellular Ca2+ levels, however, triggered cell death with a much greater proportion of the cells undergoing necrosis rather than apoptosis. A relative increase in the percentage of cells undergoing necrosis was observed in cells expressing a short hairpin RNA targeted to the SGK1 gene. Necrotic cell death evoked by cytoplasmic Ca2+ overloading was associated with persistent hyperpolarization of the inner mitochondrial membrane and a modest increase in calpain activation, but did not involve detectable caspase 3 or caspase 7 activation. The effects of cytoplasmic Ca2+ overloading on mitochondrial membrane potential were significantly reduced in cells expressing SGK1 compared with SGK1-depleted cells. Our findings indicate that store-operated Ca2+ entry regulates SGK1 expression in epithelial cells and suggest that SGK1-dependent cytoprotective signaling involves effects on maintaining mitochondrial function.

Stromal Interaction Molecule 1 (STIM1) Regulates ATP-Sensitive Potassium (KATP) and Store-Operated Ca2+ Channels in MIN6 β-Cells.

Stromal interaction molecule 1 (STIM1) regulates store-operated Ca2+ entry (SOCE) and other ion channels either as an endoplasmic reticulum Ca2+-sensing protein or when present in the plasma membrane. However, the role of STIM1 in insulin-secreting β-cells is unresolved. We report that lowering expression of STIM1, the gene that encodes STIM1, in insulin-secreting MIN6 β-cells with RNA interference inhibits SOCE and ATP-sensitive K+ (KATP) channel activation. The effects of STIM1 knockdown were reversed by transduction of MIN6 cells with an adenovirus gene shuttle vector that expressed human STIM1. Immunoprecipitation studies revealed that STIM1 binds to nucleotide binding fold-1 (NBF1) of the sulfonylurea receptor 1 (SUR1) subunit of the KATP channel. Binding of STIM1 to SUR1 was enhanced by poly-lysine. Our data indicate that SOCE and KATP channel activity are regulated by STIM1. This suggests that STIM1 is a multifunctional signaling effector that participates in the control of membrane excitability and Ca2+ signaling events in β-cells.

Interplay between ER Ca2+ Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca2+ Entry

Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca2+ signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca2+-binding protein, with Ca2+-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca2+ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca2+ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.