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Sugar beet cells maintained in the stationary phase of the batch culture cycle for 2 or more days have been shown to exhibit many of the characteristics of quiescent (G

The PD-1/PD-L1 pathway plays a critical role in the physiologic inhibition and modulation of the immune response in normal tissue. Many tumors evade immune detection and response by upregulating PD-L1 expression. Humanized monoclonal PD-1 and PD-L1 antibodies have proven as both tolerable and effective treatment in many neoplasms. Extramammary Paget disease (EMPD) is a deformative and debilitating cutaneous malignancy in which definitive treatment options are limited with high recurrence rates after surgical excision. To the best of our knowledge, there is little published information regarding EMPD and PD-L1 expression. We evaluated 18 EMPD surgical pathology cases for tumor cell and tumor-associated inflammatory (TAI) cell PD-L1 expression. We identified PD-L1 tumor cell expression in 3 (17%) of the cases: 2 of 4 invasive cases (50%) and 1 of 14 (7%) noninvasive cases. One invasive case had lymph nodal metastasis with PD-L1 tumor cell expression. The host inflammatory response intensity and PD-L1 expression were variable in cases negative for tumor cell PD-L1 expression; however, a marked inflammatory response and TAI PD-L1 expression were present in all cases positive for tumor cell PD-L1 expression. In conclusion, 1 in 14 (7%) in situ EMPD cases showed tumor cell PD-L1 expression and 2 of 4 invasive cases (50%) showed tumor cell PD-L1 expression. TAI cells were more often positive (83%) than tumor cells (17%) for PD-L1 expression.

Objectives The aim of this study was to determine the prevalence of bile cast nephropathy (BCN) in autopsied cirrhotic patients and to correlate BCN with clinical and laboratory data to direct attention to this underrecognized renal complication of liver failure. Methods We assessed 114 autopsy cases of cirrhosis for the presence of renal intratubular bile casts using Hall stain for bile. Presence of bile casts was correlated with etiology of cirrhosis, clinical and laboratory data, and histologic findings. Results Bile casts were identified in 55% of cases. The most common etiology of cirrhosis was hepatitis C virus (HCV) infection (52%), and serum creatinine ( P = .02) and serum urea nitrogen ( P = .01) were significantly higher in the Hall-positive group. Conjugated bilirubin was below 20 mg/dL in 90%, and levels below 10 mg/dL were noted in 80% of cases. Conclusions To our knowledge, this is the largest study of BCN in human subjects and a first report describing the association of BCN with HCV-related cirrhosis. We demonstrated that in the face of protracted chronic hyperbilirubinemia, bile casts are formed at much lower bilirubin levels than previously thought. Furthermore, we proposed an algorithm to assist in better identification of bile casts.

Sarcoidosis is a granulomatous disease of unknown etiology. Although sarcoidosis is a systemic disease, there appears to be a predilection for involvement of certain organs. The pulmonary system is the most commonly affected system among all racial groups. Cardiac and respiratory complications are the leading causes of death due to sarcoidosis and in certain patient populations about half of these deaths are attributed to cardiac sarcoidosis. There are few autopsy case reports of cardiac sarcoidosis with minimal respiratory involvement making this case report relevant to the importance of the recognition and awareness of this entity. Acad Forensic Pathol. 2018 8(2): 407-415

The aim of this study was to determine the prevalence of bile cast nephropathy (BCN) in autopsied cirrhotic patients and to correlate BCN with clinical and laboratory data to direct attention to this underrecognized renal complication of liver failure.We assessed 114 autopsy cases of cirrhosis for the presence of renal intratubular bile casts using Hall stain for bile. Presence of bile casts was correlated with etiology of cirrhosis, clinical and laboratory data, and histologic findings.Bile casts were identified in 55% of cases. The most common etiology of cirrhosis was hepatitis C virus (HCV) infection (52%), and serum creatinine ( P = .02) and serum urea nitrogen ( P = .01) were significantly higher in the Hall-positive group. Conjugated bilirubin was below 20 mg/dL in 90%, and levels below 10 mg/dL were noted in 80% of cases.To our knowledge, this is the largest study of BCN in human subjects and a first report describing the association of BCN with HCV-related cirrhosis. We demonstrated that in the face of protracted chronic hyperbilirubinemia, bile casts are formed at much lower bilirubin levels than previously thought. Furthermore, we proposed an algorithm to assist in better identification of bile casts.

This paper reports the ways that the differences in leaf senescence are related to grain filling, grain yield, and the dynamics of cytokinins (CKs) in the top three leaves of four field-grown new plant type (NPT) rice, a tropical japonica developed at the International Rice Research Institute, Philippines, to increase the yield potential of rice. The chlorophyll content in leaves decreased from flowering to maturity in all the NPT lines, whereas the grain filling percentage was higher in the fast-senescing NPT line than in slow-senescing NPT line. Grain yield was positively correlated with senescence in the flag leaf. Rapid changes in the CK levels were recorded in the leaves of the fast-senescing line, whereas the CK levels were relatively stable in leaves of the slow-senescing line, suggesting that the dynamics of CKs in the fast-senescing line are vital for fast senescence. There were no significant changes in bioactive CKs, CK O-glucosides (storage CKs), and cis-zeatin derivatives in different leaves of the slow-senescing NPT line between 0 and 3 weeks after flowering, suggesting that the content of these CKs is relatively stable during grain filling. A progressive increase in levels of bioactive CKs was positively correlated with gradual accumulation of CK N-glucosides (inactive CKs) in the top three leaves of the slow-senescing NPT line, whereas the decrease of bioactive CKs in the flag leaf of the fast-senescing line was accompanied by accumulation of CK O-glucosides. These results suggest that there is a higher rate of biosynthesis and/or import of bioactive CKs as well as their turnover which may favor delay of leaf senescence in the slow-senescing NPT line.

the myriad of intracellular signaling pathways involving intracellular Ca2+ means Ca2+ plays a role in everything from cellular metabolism to cell death. In heart muscle Ca2+ is primarily known for its role in cellular contraction. Regulation of cardiac muscle contractility is achieved through

We examined the modulation of the cardiac L-type Ca(2+) channel (LTCC) by the regulatory protein sorcin and tested the hypothesis that modulation occurred by direct interaction. Whole-cell patch-clamp recordings were made on native rabbit ventricular myocytes and HEK 293 cells expressing cardiac alpha(1C) subunits. In ventricular cells, sorcin increased peak current when using either Ca(2+) or Ba(2+) as charge carriers. In HEK 293 cells, sorcin increased peak current density when using Ba(2+) as a charge carrier but not when using Ca(2+). In ventricular myocytes, current inactivation (tau(fast), in ms) was slowed by sorcin with Ca(2+) as the charge carrier, whilst in the presence of Ba(2+) it was enhanced. In HEK 293 cells, sorcin significantly enhanced tau(fast), but no significant change was observed with Ba(2+). This trend was mimicked by the truncated peptide, sorcin Ca(2+)-binding domain, which lacks the N-terminal domain. These data suggest that sorcin interacts with LTCC via its C-terminal domain, which alters current magnitude and tau(fast). These effects appear to be influenced by the prevailing experimental conditions.

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root-knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest-resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect-host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.

Age and hypertension contribute significantly to cardiac morbidity and mortality, however the importance of each during the progression of hypertrophy is unclear. This investigation examined the effect of age and hypertension on Ca(2+) handling in rat ventricular myocytes by comparing a genetic model of hypertension and cardiac hypertrophy (spontaneously hypertensive rat, SHR) with its normotensive control (Wistar-Kyoto rat, WKY) at 5 and 8 months of age. Experiments were performed on single left ventricular myocytes isolated from SHR or WKY hearts. Intracellular Ca(2+) was measured optically using fura-2 or fluo-3. SHR myocytes had a significantly larger cell width and volume and a significantly decreased cell length/width ratio at 5 and 8 months compared to normotensive controls. Age had no effect on cell length, width, volume or the length/width ratio. Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) content and contraction amplitude were unaffected by age or hypertrophy. However at 8 months the contribution of the SR to Ca(2+) uptake during relaxation decreased, with a concomitant increase in the contribution of Na(+)/Ca(2+) exchanger (NCX) function to relaxation, in SHR and WKY myocytes. The incidence of non-synchronous SR Ca(2+) release decreased with age but not hypertrophy in SHR and WKY myocytes. These results show that the changes in Ca(2+) handling observed during progression of mild hypertrophy in SHR are the same as those that occur during ageing in normotensive control animals and can, therefore, be ascribed to maturation rather than hypertrophy.

Changes in cellular calcium (Ca(2+)) handling are thought to underlie the altered contraction that occurs during cardiac hypertrophy and failure. Recent work has highlighted the importance of t-tubules in the control of intracellular Ca(2+). The present study was performed to investigate whether changes in the distribution of I (Ca) between the surface and t-tubule membranes might contribute to the altered Ca(2+) handling observed during compensated hypertrophy in the spontaneously hypertensive rat (SHR). Experiments were performed on ventricular myocytes isolated from 5-month-old SHR and normotensive Wistar-Kyoto (WKY) control rats. Osmotic shock using formamide was used to disrupt the t-tubular system and the whole-cell patch clamp technique used to monitor I (Ca) in the presence and absence of t-tubules. Membrane capacitance and I (Ca) were greater in control SHR than WKY myocytes; following detubulation, cell capacitance and I (Ca) both decreased and were no longer significantly different in the two cell types. The density of I (Ca) was not significantly different in control SHR and WKY cells or in detubulated myocytes from the two species. These data suggest that the distribution of I (Ca) is unchanged in SHR myocytes compared to WKY controls; I (Ca) density in the t-tubules was 1.2-fold greater than in the sarcolemma in both strains. These data also imply that the increase in surface area in SHR myocytes is due principally to an increase in t-tubular area, which is accompanied by an approximately equivalent increase in I (Ca), so that the density of I (Ca) at the cell surface and in the t-tubules remains the same. These changes would be expected to retain cell function and synchronicity of Ca(2+) release in the SHR at this stage of compensated hypertrophy.

Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+from the sarcoplasmic reticulum (SR), and Ni2+(10 mM) was used to inhibit Na+/Ca2+exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+transient amplitude and SR Ca2+content and less Ca2+extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+content and hence Ca2+transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.

A global and transient rise of intracellular Ca2+ (Ca2+i) is central to the operation of pump-leak coupling in the frog early distal tubule (EDT). The endoplasmic reticulum (ER) is the site of this Ca2+ release and reuptake; however, it is likely that other intracellular pools, such as mitochondria, also contribute to cellular Ca2+ homeostasis. The present study was performed to seek evidence of mitochondrial Ca2+ transport in the frog EDT. Experiments were performed on isolated and permeabilized EDT segments from the frog kidney loaded with the low-affinity, Ca2+-sensitive fluorescent indicator, mag-fura-2. Ca2+ uptake in the absence of SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) activity (inhibition by 2,5-di-t-butyl hydroquinone, TBQ) was evident at a bath [Ca2+] of 1 microm, but not at 200 nm, in the presence of ATP. This uptake was sensitive to the protonophore FCCP and the ATP-synthase inhibitor oligomycin. Ca2+ uptake was also stimulated by respiratory substrates; this uptake was enhanced by oligomycin and reversed by the application of FCCP. These findings provide the first evidence of mitochondrial Ca2+ transport in renal tubules, which appears to occur via a low-affinity pathway and which will act as a physiological Ca2+ buffer, protecting the cell from large increases in Ca2+i.

The early distal tubule (EDT) of the frog nephron, similar to the thick ascending limb in mammals, mediates the transepithelial absorption of NaCl. The continued absorption of NaCl in the face of varying Na+load is maintained by coordination of the activity of ion-transporting proteins in the apical and basolateral membranes, so-called pump-leak coupling. Previous studies identified intracellular Ca2+, originating from an intracellular Ca2+store, as playing a key role in pump-leak coupling in the EDT (Cooper GJ, Fowler MR, and Hunter M. Pflügers Arch 442: 243–247, 2001). The purpose of the experiments described in this paper was to identify the intracellular Ca2+storage pools in the renal diluting segment. Store Ca2+movements were monitored by the fluorescence of mag-fura 2 in permeabilized segments of frog EDTs. The presence of both ATP and Ca2+was required to maintain store Ca2+content. Removal of either of these substrates resulted in a passive leak of Ca2+from the stores. The uptake of Ca2+into the store was sensitive to the SERCA inhibitor 2,5-di( tert-butyl) hydroquinone, whereas Ca2+release from the store was stimulated by IP3but not cADPR. Store Ca2+was insensitive to the mitochondrial ATP synthase inhibitor oligomycin, and, under conditions that energized Δψm, the complex 1 inhibitor rotenone and the protonophore FCCP. Ionomycin was able to mobilize store Ca2+following exposure to IP3. These results suggest that the endoplasmic reticulum is a dominant Ca2+store in the frog EDT. A second pool, sensitive to ionomycin but not IP3, may overlap with the IP3-sensitve pool. The data also rule out any contribution by mitochondria to EDT Ca2+cycling.

The activities of transport mechanisms in epithelial cells are generally coordinated in order to minimise disturbances in cellular ion content and volume. Furosemide, a potent inhibitor of transport in the renal diluting segment, up-regulates apical K+ channel activity following the release of calcium from intracellular stores. The signal pathway between furosemide application and this calcium release is not known. Single early distal tubule segments from frog kidney were permeabilised with saponin in order to monitor calcium levels within cytoplasmic stores using the calcium-sensitive dye, mag-fura. The uptake (or release) of calcium to (or from) stores was initiated by adding agents to the bath solution, which is in direct contact with the intracellular organelles. ATP promoted calcium uptake into stores, whereas ATP removal led to a slower, spontaneous calcium release. Following loading, calcium stores could be rapidly depleted by inositol 1,4,5-trisphosphate (IP3), but not ryanodine. Calcium release was evident upon lowering the "intracellular" chloride concentration from 12 to 4 mM, equivalent to the fall in chloride induced by furosemide in intact cells. These results suggest that intracellular chloride may function as a second messenger, mediating cross-talk between the apical membrane and intracellular calcium stores.

The co-transformation of a single plant genome with two independent T-DNA regions provides opportunities for genetic separation in subsequent generations. In an effective strategy, co-delivery events must form a high proportion of the total transformed population. In this study, using the model plant species tobacco (Nicotiana tabacum), it was shown that the frequency of co-transformation within a given To population could be as high as 100% and this was found to be dependent, at least in part, on designing the plasmid vectors so that the kbp size of the first selected T-DNA region was >2-fold that of the designated T-DNA region for co-transfer. Overall, 40-50% of To lines demonstrated the capacity for segregational separation of co-transformed T-DNA regions. Hence, the estimate of the required number of total transformants for such an independent strategy may seem to be as little as 2-fold that for a conventional, single T-DNA strategy, but we strongly temper such estimates with indications that high co-transformation frequencies may be associated with a higher incidence of linkage. In this co-transformation study we used a single (Agrobacterium) strain system in which a single binary plasmid contained either two or three T-DNA regions, each with a selectable marker. This arrangement could reveal that 'read-through' events within the Agrobacterium cells, resulting in the co-transfer of adjacent T-DNA regions as a single linked unit, accounted for up to 20% of co-transformed plant lines. Such read-through co-delivery appeared to be more frequent from the 'supervirulent' EHA101 A. tumefaciens strain, compared to the 'ordinary' LBA4404 strain. By using the binary plasmid with three selectable T-DNA regions, we have been able to consider the frequency of co-integration of a third independent T-DNA within a T0 subpopulation of co-transformants. This was found to be higher than expected. These observations were applied to the co-transfer of (unwanted) plasmid backbone sequences and showed that screening against such sequences may add a significant factor in achieving the desired, final genotype.

Biological scientists are eagerly confronting the challenge of understanding the regulatory mechanisms that control the cell division cycle in eukaryotes. New information will have major implications for the treatment of growth-related diseases and cancer in animals. In plants, cell division has a key role in root and shoot growth as well as in the development of vegetative storage organs and reproductive tissues such as flowers and seeds. Many of the strategies for crop improvement, especially those aimed at increasing yield, involve the manipulation of cell division. This review describes, in some detail, the current status of our understanding of the regulation of cell division in eukaryotes and especially in plants. It also features an outline of some preliminary attempts to exploit transgenesis for manipulation of plant cell division.

Sugar beet cells grown in liquid suspension culture entered the stationary phase on day 17 of the batch culture cycle as a consequence of nutrient depletion. Between days 17 and 19 the cells exhibited a number of marked changes in RNA and protein metabolism characteristic of entry into a quiescent or G0 state. Protein synthesis declined considerably and very few polypeptides incorporated sufficient radioactively labelled methionine to be detected by fluorography following polyacrylamide gel electrophoresis (SDS-PAGE). There was a marked decrease in protein half life during the stationary phase and this was accompanied by a rise in protease activity. RNA synthesis also decreased to

Sugar beet cells maintained in the stationary phase of the batch culture cycle for 2 or more days have been shown to exhibit many of the characteristics of quiescent (G0) cells. When such cells were subcultured into fresh medium they progressed through a period of DNA synthesis to a highly synchronised first division, 6 days after subculture. The onset of DNA synthesis and cell division were each delayed by 2 days relative to the timing of the events when the cells were subcultured immediately before entry into the stationary phase. The regulation of gene expression during this extended transition from the G0 phase back to the cell division cycle was investigated. The cell division cycle-related genes Bvcdc2, Betvu;CycA2, Arath;CycB1;1 histone H4 and Bvcrk1 (a novel cdc2-like gene) showed widely differing patterns of expression. Bvcdc2 transcripts were present at low levels in quiescent cells whereas crk1, cyclin and histone transcripts were not detectable. Expression of both Bvcrk1 and Betvu; CycA2 was induced within 1 h after subculture into fresh medium, whereas histone H4 gene expression was not detectable for 24 h and showed a marked increase between 24 and 48 h. B-type cyclin transcripts were not detectable until more than 48 h after subculture. The addition of either sucrose or MS macronutrients to quiescent sugar beet cells was not sufficient to re-initiate cell division but both medium components were able to stimulate the expression of the two ‘early’ genes (Betvu;CycA2 and Bvcrk1) within 6 h. Furthermore, although the sugar beet cells were habituated, i.e. they were routinely grown without added plant growth regulators, treatment of quiescent cells with IAA and kinetin also induced expression of Betvu;CycA2 and Bvcrk1 without subsequent cell division.

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S-adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence (BvcycII).

Early CHARMM all-atom force fields showed transition temperatures well above the experimental value of 315K. We simulated 100 ns for DPH and TMA-DPH dyes in DPPC lipid bilayers at atmospheric pressure and temperatures from 300-330K in 5K increments. Shifts were observed between 305K and 315K in area-per-headgroup, bilayer thickness, lipid-tail order parameters, and steady state dye fluorescence anisotropy. Dye effects on these membrane properties were negligible.

Background An efficient method for the identification of medicinal plant products is now a priority as the global demand increases. This study aims to develop a DNA-based method for the identification and authentication of plant species that can be implemented in the industry to aid compliance with regulations, based upon the economically important Hypericum perforatum L. (St John’s Wort or Guan ye Lian Qiao). Methods The ITS regions of several Hypericum species were analysed to identify the most divergent regions and PCR primers were designed to anneal specifically to these regions in the different Hypericum species. Candidate primers were selected such that the amplicon produced by each species-specific reaction differed in size. The use of fluorescently labelled primers enabled these products to be resolved by capillary electrophoresis. Results Four closely related Hypericum species were detected simultaneously and independently in one reaction. Each species could be identified individually and in any combination. The introduction of three more closely related species to the test had no effect on the results. Highly processed commercial plant material was identified, despite the potential complications of DNA degradation in such samples. Conclusion This technique can detect the presence of an expected plant material and adulterant materials in one reaction. The method could be simply applied to other medicinal plants and their problem adulterants.

A strategy has been identified for the production of a low soil tare sugar beet by genetic manipulation of the table beet. This approach will involve increasing the sucrose storage capacity of the table beet by manipulating cell division in the cambial rings of the storage organ. Research aimed at the isolation of suitable cell division regulatory genes and storage organ specific promoter sequences is described. Sugar beet cdc 2 and cyclin sequences have been isolated by PCR techniques. Two root specific cDNA sequences have been characterized; one belongs to a group of hybrid proline-rich/hydrophobic proteins and the other resembles a gene induced during potato tuberisation.

Cell division and the cell cycle are fundamental processes that are well, though not absolutely, conserved in all eukaryotes. Suspension cultured plant cells that can be synchronized offer the ideal experimental system in which to study the events and controls of the plant cell division cycle. As would be expected, the events during and controls of the plant cell division cycle appear to be very similar to those in other multicellular eukaryotes; there are, however, some important differences in both the processes during the cell division cycle and the controls that act on the cell division cycle. These differences may reflect the unique physical processes that accompany cell division in plants, the unique developmental patterns of plants or the differing signaling pathways utilized by plants to coordinate growth, division, and development. Keywords: CDK; cell cycle; cell division; cell growth; cell suspension; cyclin

Modulation of the L-type Ca2+ channel (LTCC) by sorcin was investigated by measuring the L-type Ca2+ current (I Ca,L) in isolated rabbit ventricular myocytes using ruptured patch, single electrode voltage clamp in the absence of extracellular Na+. Fifty millimolars EGTA (170 nM Ca2+) in the pipette solution buffered bulk cytoplasmic [Ca2+], but retained rapid Ca2+-dependant inactivation of I Ca,L,. Recombinant sorcin (3 μM) in the pipette significantly slowed time-dependant inactivation (τ fast: 8.8 ± 0.9 vs. 15.1 ± 1.7 ms). Sorcin had no significant effect on I Ca,L, after inhibition of the sarcoplasmic reticulum (SR). Using 10 mM 1,2-bis(o-N,N,N′,N′-tetraacetic acid (170 nM Ca2+), I Ca,L inactivation was then determined by a Ca2+ -independent, voltage-dependant process. Under these conditions, 3 μM sorcin speeded up inactivation. A similar effect was observed by substitution of Ca2+ with Ba2+. Down-regulation of endogenous sorcin to 27 ± 7% using an RNAi adenoviral vector slowed inactivation of I Ca,L by ∼42%. The effects of sorcin on voltage-dependant inactivation were mimicked by a truncated form of the protein containing only the Ca2+-binding domain. This data is consistent with two independent actions of sorcin on the LTCC: (1) slowing Ca2+-dependant inactivation and (2) stimulating voltage-dependant inactivation. The net effect of sorcin on the time-dependent inactivation of I Ca,L was a balance between these two effects. Under normal conditions, sorcin slows I Ca,L inactivation because the effects of Ca2+-dependant inactivation out-weigh the effects on voltage-dependant inactivation.

The nuclear ribosomal internal transcribed spacer (ITS) sequences of eight Hypericum species were used to design H. perforatum-specific PCR primers by identification of short "microcode" sequences characteristic of the target species. These were tested with three vouchered H. perforatum DNA samples and eight samples from other species within the Hypericum genus. The most efficient primer combination, FO2 and HRI-S, amplified the genomic DNA from all three H. perforatum samples but not from any of the others apart from H. delphicum. The primer pairing was then tested against seven commercially available ornamental varieties of Hypericum; a positive result was obtained only with the H. perforatum sample. Three consumer products retailed as "St. John's wort" herbal remedies were sampled, two of which gave a positive result for H. perforatum. The assay was sensitive enough to detect 0.75 ng H. perforatum present as just 0.1 % of the total DNA. This method has the potential to be replicated in other plant species and presents a novel use for DNA barcoding data.

Excitation–contraction (E–C) coupling in cardiac muscle is the transduction mechanism linking the action potential to cell shortening which occurs in response to a transient rise in intracellular calcium (Ca2+i). Activation of the Ca2+ current (ICa,L, the substrate of which is the L-type Ca2+ channel (LTCC) or dihydropyridine receptor (DHPR)) triggers a larger release of Ca2+ from the sarcoplasmic reticulum (SR) store via Ca2+-induced Ca2+ release (CICR) due to activation of the SR Ca2+ release channel, the ryanodine receptor (RyR), on the SR membrane. Experimental evidence strongly supports ‘local control’ of Ca2+ release where a cluster of RyRs are juxtaposed with DHPRs forming a couplon or Ca2+ release unit (CRU) that functions autonomously. The voltage sensitivity of DHPR activity and the progressive recruitment of couplons is the cornerstone of graded Ca2+ release and contractility in cardiac tissue. Although the relationship between trigger Ca2+ and SR Ca2+ release has received much attention, critical questions still remain about the nature of the coupling between LTCC and RyR. In particular, elementary Ca2+ release events (Ca2+ sparks) can be triggered with very high probability during an action potential (AP) but at the peak of the AP the probability of DHPR openings is very much lower than that of Ca2+ sparks (Inoue & Bridge, 2003). Therefore, more than one DHPR must be involved in triggering SR Ca2+ release but it is not clear exactly how many DHPR channels reside in a couplon and how the numbers of channels and their activity modulates SR Ca2+ release. In a recent paper in The Journal of Physiology, Polakova et al. (2008) significantly advance our understanding of the local control process in a complex but powerful study, by demonstrating that a significant number of DHPRs are present in a couplon (20–40) and that at least eight of these channels must be open simultaneously in order to trigger SR Ca2+ release Spontaneous and triggered local release events have been intensively studied since the first description of Ca2+ sparks by Cheng et al. in 1993 (Cheng et al. 1993). An advance on the Ca2+ spark methodology was demonstrated by Song et al. (1998) where a low-affinity Ca2+-sensitive fluorescent probe was combined with high concentrations of EGTA which reported SR Ca2+ release from a couplon. This release activity was termed a Ca2+ spike. Ca2+ spikes are a more appropriate approach where SR Ca2+ release flux is important than measuring Ca2+ sparks, since the Ca2+ spark signal also contains components not directly associated with SR Ca2+ release flux. This is especially evident in the rising and decay phases of Ca2+ sparks, which are intrinsically much slower than Ca2+ spikes. Ca2+ spikes are therefore able to report the latency of Ca2+ release quite faithfully (Zahradnikova et al. 2007). The Ca2+ spike approach involves the use of EGTA in the cell (introduced via the patch pipette), which prevents diffusion of the Ca2+ signal, thereby filtering out global cytosolic changes in Ca2+ whilst the low-affinity indicator reports the large Ca2+ release flux. Polakova and co-workers apply this methodology to rat ventricular myocytes where the Ca2+ spike signal consists of two components: a dominant component attributable to SR Ca2+ release flux, which is manifest as a rapid and transient spike, and a minor component representing the integral of Ca2+ release from the SR. Song et al. (1998) describe this as a ‘pedestal’ since during a voltage pulse this component of the signal remains elevated above baseline. In addition to this powerful methodology, Polakova and colleagues utilize a clever voltage clamp protocol that permits control not only of the number of channels triggered to open but also the driving force acting on those channels. Channels were activated with minimal Ca2+ flux by a depolarizing prepulse to Erev of ICa,L (+60 mV). Rapid repolarization increased the driving force for Ca2+ entry in a temporally synchronized manner and in this aspect the protocol is superior to traditional step depolarizations since channel open probability and driving force would otherwise change in opposite directions. The number of channels activated was controlled by the time spent at +60 mV (1.5 or 5 ms). Line scanning confocal microscopy monitored the occurrence of Ca2+ spikes. A strong component of the research paper was the use of mathematical modelling to describe the stochastic nature of Ca2+ release events (probability, latency) and it is from this computational analysis that many of the significant conclusions of the paper are drawn. The experimental procedure described above was used by the group to demonstrate that the number of DHPRs activated in the couplon is a significant factor in triggering Ca2+ release. Varying the number of channels activated (controlled by the prepulse to +60 mV) affected spike probability and latency. Spike probability was higher and latency was shorter with larger numbers of activated channels. Where maximal numbers of channels were activated compared to only a fraction of channels (5 ms versus 1.5 ms prepulse), the difference between the spike probability was smallest at –40 mV. This repolarization voltage does not inhibit channel reopenings during the tail current since this potential is not sufficiently hyperpolarized to fully inactivate the channels. Therefore, the differences in latency between the two fractions of activated DHPRs were largest at –40 mV since larger numbers of channel reopenings contribute to increasing the synchrony of SR release. Given that these results demonstrate the importance of the number of DHPR channels for SR release, an examination of lengthening the channel open time with the agonist BayK 8644 was performed. This rendered spike properties independent of the repolarization potential, suggesting that, in addition to the numbers of channels open, the channel open time may also be an important factor in the coupling of SR release, a situation that has physiological relevance since it mimics modulation by β-adrenergic agonists in cardiac tissue. However, this result also describes a paradox in the data reported by this group. The analysis carried out by Polakova and colleagues was applied to the distribution of spike latencies. Latency may be interpreted simply as the synchrony of Ca2+ release from the couplon and this synchrony is regulated by the open probability and number of DHPRs in the couplon. As more channels are recruited (5 ms versus 1.5 ms prepulse) synchrony increases, i.e. more spikes occur with shorter latencies. The paradox rests in that prolonging channel openings with BayK 8664, synchrony (latency) was not increased and in fact it was longer than control. The interpretation of these data is that the number of active channels is a dominant feature in the control of SR Ca2+ release flux and not the length of channel openings per se. However, these data are inconsistent with other reports that demonstrate increased synchrony with β-adrenergic stimulation (which also has the effect of increasing channel open times (Song et al. 2001)). Both the application of BayK 8664 and β-adrenergic stimulation shift the voltage dependence of DHPRs in favour of activation at more negative voltages. This may have the effect of (1) recruiting more channels in the couplon which would explain the increased synchrony observed by other groups or (2) the effects of lengthening DHPR open times may not be discernable during an activating prepulse to +60 mV, and explains the paradox reported by the Polakova group. SR load has a significant impact of SR release flux which may also have contributed to this result. The analysis used by Polakova and co-workers enabled description of the coupling fidelity of SR release (i.e. the ability of a single DHPR to trigger SR Ca2+ release). The equation used to explain this includes the number of DHPRs in a couplon, channel open probability and the number of channels that open sequentially (together) (see eqn 4 in the paper). The probability and latency distribution were modelled to derive estimations of these parameters. Significantly, this demonstrated that the coupling fidelity is very low (0.15 at –120 mV) and thus the only way that SR release can occur with high probability (i.e. to mimic the experimental data) is if multiple channels open simultaneously in a couplon. It is from this analysis that the group surmise that at least eight channels must be open in a couplon during activation to trigger SR release and that a couplon contains between 20 and 40 DHPRs. This represents the key advance to our understanding of local control of E–C coupling. The power of this analysis lies in that it quantitatively describes the numbers of DHPRs of a total couplon population that are required to be active in order to trigger SR release and provides a paradigm for explaining the responses to adrenergic stimulation (Song et al. 2001), during pathological remodelling and variable E–C coupling gain. Each of these situations may result in the numbers and activity of DHPRs being modified and explains the capacity for physiological regulation by the local control mechanism. In summary the paper by Polakova and colleagues represents a significant advance in our understanding of the local control mechanism of E–C coupling. The number of channels in a couplon and the minimum number required to open to trigger SR flux are likely to be modified by adrenergic stimulation, regional location, species and during cardiac disease. Application of their approach to these situations is likely to provide a fruitful area for future investigation.

We examined the modulation of the cardiac L-type Ca(2+) channel (LTCC) by the regulatory protein sorcin and tested the hypothesis that modulation occurred by direct interaction. Whole-cell patch-clamp recordings were made on native rabbit ventricular myocytes and HEK 293 cells expressing cardiac alpha(1C) subunits. In ventricular cells, sorcin increased peak current when using either Ca(2+) or Ba(2+) as charge carriers. In HEK 293 cells, sorcin increased peak current density when using Ba(2+) as a charge carrier but not when using Ca(2+). In ventricular myocytes, current inactivation (tau(fast), in ms) was slowed by sorcin with Ca(2+) as the charge carrier, whilst in the presence of Ba(2+) it was enhanced. In HEK 293 cells, sorcin significantly enhanced tau(fast), but no significant change was observed with Ba(2+). This trend was mimicked by the truncated peptide, sorcin Ca(2+)-binding domain, which lacks the N-terminal domain. These data suggest that sorcin interacts with LTCC via its C-terminal domain, which alters current magnitude and tau(fast). These effects appear to be influenced by the prevailing experimental conditions.

Accurate quantitative assays are required for enforcing food labelling procedures and preventing food ingredient contamination, misdescription and fraud. Simplex and duplex TaqMan real-time PCR systems have been tested for the identification and quantification of DNA in meat, milk and cheese. DNA was isolated from meat and cheese using a standard CTAB protocol and from milk using a Promega Wizard Magnetic kit and purified by Qiagen silicon spin columns. High quality DNA isolated from beef mince was used for standard curve construction in the TaqMan real-time PCR assay using a bovine-specific primer pair for the mitochondrial cytb gene and a FAM-labelled mammalian-specific cytb probe. The real-time PCR assay can quantitatively detect as little as 35 pg bovine DNA and showed no cross-reaction with ovine, caprine or porcine DNA. The system has been successfully used to measure bovine DNA in fresh and processed meat, milk and cheese, and will prove useful for bovine species identification and quantitative authentication of animal-derived products.

A global and transient rise of intracellular Ca2+ (Ca2+i) is central to the operation of pump-leak coupling in the frog early distal tubule (EDT). The endoplasmic reticulum (ER) is the site of this Ca2+ release and reuptake; however, it is likely that other intracellular pools, such as mitochondria, also contribute to cellular Ca2+ homeostasis. The present study was performed to seek evidence of mitochondrial Ca2+ transport in the frog EDT. Experiments were performed on isolated and permeabilized EDT segments from the frog kidney loaded with the low-affinity, Ca2+-sensitive fluorescent indicator, mag-fura-2. Ca2+ uptake in the absence of SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) activity (inhibition by 2,5-di-t-butyl hydroquinone, TBQ) was evident at a bath [Ca2+] of 1 microm, but not at 200 nm, in the presence of ATP. This uptake was sensitive to the protonophore FCCP and the ATP-synthase inhibitor oligomycin. Ca2+ uptake was also stimulated by respiratory substrates; this uptake was enhanced by oligomycin and reversed by the application of FCCP. These findings provide the first evidence of mitochondrial Ca2+ transport in renal tubules, which appears to occur via a low-affinity pathway and which will act as a physiological Ca2+ buffer, protecting the cell from large increases in Ca2+i.

Objective: Recent work has suggested that Na + /Ca 2+ exchange (NCX) and L-type Ca 2+ current (ICa) are located predominantly in the ttubules of cardiac ventricular myocytes, which therefore represent a microdomain for the regulation of intracellular Na + (Nai) and Ca 2+ (Cai). The aim of this study was to investigate the role of the t-tubules in the response of Cai and contraction to interventions that alter the transsarcolemmal Na + gradient. Methods: Enzymatically isolated and detubulated Wistar rat ventricular myocytes were investigated using fluorescence microscopy and optical detection of cell length. Results: In unstimulated cells, spontaneous contractile activity increased when extracellular [Na + ] was decreased or strophanthidin (100 AM) was added to the bathing solution, but the increase was significantly smaller in detubulated cells than in control cells. In electrically stimulated cells, strophanthidin increased Nai to a similar extent in normal and detubulated cells, although the associated increase in Ca 2+ transient amplitude and contraction were significantly smaller in detubulated cells. Similarly, tetrodotoxin (TTX, 10 AM) attenuated the Ca 2+ transient and contraction less in detubulated than in control cells. Increasing stimulation rate (0.05–1 Hz) caused little change or a small increase in contraction amplitude in control cells, but a significant decrease in contraction amplitude in detubulated cells, although the change of Nai caused by increasing stimulation rate from 0 to 1 Hz was not significantly different in the two cells types. Conclusion: It is concluded that although some Na/K ATPase, NCX and Na + channel activity is present on the surface membrane, the t-tubules play a major role in the modulation of contraction via NCX, allowing changes of the transsarcolemmal Na + gradient to be translated into changes of Cai.

Cell growth and division are integrated with a programme of differentiation to determine the form of the plant body. In recent years the molecular controls of cell division and growth have been elucidated. Progression through the cell cycle is controlled by a family of serine/threonine protein kinases termed CDKs (cyclin-dependent kinases) that act in association with regulatory proteins termed cyclins. Specific CDKs associate with specific cyclins in order to bring about both the gene expression changes and the physical modifications that are necessary to complete the cell cycle. The nature and interactions of CDKs and cyclins have been studied in detail in mammalian and yeast cells but there is still relatively little information about such systems in plants. Much of the information is drawn by inference. Little is known about the coupling of cell division with cell growth and the integration of these processes into development.

A novel gene (RS2) has been isolated from a Beta vulgaris (cv. Regina) cDNA library. The expression of this gene was enhanced in the mature storage organ as compared to leaf tissue. The protein encoded by this gene was found to be alanine- and glutamic acid-rich and it resembles members of the latex allergen Hev b 5 family.

The sucrose storage capacity of the sugar beet, Beta vulgaris, storage organ has been related to its anatomy. A transverse section of the storage organ reveals a pattern of concentric rings which is caused by the alternation of vascular zones with parenchymatous zones. Sucrose is transported to the storage organ via the phloem and moves laterally to the adjacent parenchymatous tissues, where it is stored in the cell vacuoles. The storage organ of sugar beet has 12–15 cambia, the divisions of the 6 inner rings provide the cells which make up some 75% of the storage root while the outer rings (9 and above) make almost no contribution to the bulk of the storage root. It has been found that the small parenchymatous cells close to the phloem accumulate sucrose to a higher concentration than that achieved by large (older) cells remote from the phloem. Hence, it is predicted that for optimum sucrose accumulation the storage organ should have a large number of parenchymatous zones, each of which offers a short diffusion path from the phloem to predominately small storage cells. In order to manipulate sugar beet development toward this optimum it is necessary to have an understanding of the processes that regulate cell division, cell expansion and differentiation. Towards this end we have begun to isolate and characterise cell division cycle related genes from sugar beet and we have developed a cell suspension culture system for analysis of their roles (Elliott et al. 1996).

to the editor: The above Viewpoint article summarized and integrated evidence in support of a key role for localized Ca2+ in the regulation of cardiac excitation-contraction (E-C) coupling. The central notion of this is that by virtue of the speed of Ca2+ release and/or the trapping of Ca2+ by