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The dermis of sea cucumbers is a catch connective tissue or mutable connective tissue that exhibits large changes in mechanical properties. A stiffening protein, tensilin, has been isolated from the sea cucumber Cucumaria frondosa. We purified a similar protein, H-tensilin, from Holothuria leucospilota, which belongs to a different family to C. frondosa. H-tensilin appeared as a single band with an apparent molecular mass of 34 kDa on SDS-PAGE. No sugar chain was detected. Tryptic fragments of the protein had homology to known tensilin. H-tensilin aggregated isolated collagen fibrils in vitro in a buffer containing 0.5 mol l–1 NaCl with or without 10 mmol l–1 Ca2+. The activity of H-tensilin was quantitatively studied by dynamic mechanical tests on the isolated dermis. H-tensilin increased stiffness of the dermis in the soft state, induced by Ca2+-free artificial seawater, to a level comparable to that of the standard state, which was the state found in the dermis rested in artificial seawater with normal ionic condition. H-tensilin decreased the energy dissipation ratio of the soft dermis to a level comparable to that of the standard state. When H-tensilin was applied on the dermis in the standard state, it did not alter stiffness nor dissipation ratio. The subsequent application of artificial seawater in which the potassium concentration was raised to 100 mmol l–1 increased stiffness by one order of magnitude. These findings suggest that H-tensilin is involved in the changes from the soft state to the standard state and that some stiffening factors other than tensilin are necessary for the changes from the standard to the stiff state.
Echinoderms show fast and large changes in the mechanical properties of their connective tissues (Wilkie, 2002; Wilkie et al., 2004). These unique tissues include catch connective tissue (mutable connective tissue) and contractile connective tissue. Catch connective tissue changes its passive mechanical properties but is not contractile in the physiological condition, unlike the contractile connective tissue of crinoids (Birenheide and Motokawa, 1996; Birenheide et al., 2000; Motokawa et al., 2004). Catch connective tissue was regarded as one of the main characteristics of members of the phylum Echinodermata and was believed to have played a key role in the success of this phylum (Motokawa, 1988). This tissue is mostly composed of extracellular materials such as collagen fibres and proteoglycans. Although the changes in the mechanical properties have been attributed to the changes in extracellular materials, the molecular mechanism of the changes is still poorly understood. The changes in mechanical properties are controlled through nerves. Neuropeptides that stiffen or soften the holothurian catch connective tissue have been found in sea cucumbers (Birenheid et al., 1998; Inoue et al., 1999). The scenario so far proposed is that nerves control the secretory activities of some cells whose secretion(s) causes changes in the number of cross-links between extracellular macromolecules and thus stiffens or softens catch connective tissues (Wilkie et al., 2004).
The holothurian dermis is a typical catch connective tissue that changes its mechanical properties in response to various stimuli. These include mechanical, photic, electrical and chemical stimulation (Motokawa, 1981; Motokawa, 1984b). Chemical stimulation by artificial seawater (ASW) with altered ionic composition was often used to induce stiffness changes because of its sustained effect and availability. ASW with an elevated concentration of K+ (KASW) induces stiffening (Motokawa, 1984c; Trotter and Chino, 1997; Motokawa and Tsuchi, 2003), whereas ASW from which Ca2+ is removed (Ca2+-free ASW) induces softening (Motokawa and Hayashi, 1987; Trotter and Koob, 1995; Trotter and Chino, 1997; Koob et al., 1999; Szulgit and Shadwick, 2000; Motokawa and Tsuchi, 2003). These media induce rapid stiffening or softening that reaches the maximum or minimum stiffness value within 10 min and that value is maintained for not less than 1 h. The changes in the ionic environment are thought to have effects on cellular elements in the dermis rather than directly on its extracellular matrix. KASW is likely to stiffen the dermis through a membrane depolarization because the Triton-extracted dermis does not stiffen in response to KASW (Motokawa, 1994). The removal of Ca2+ is believed to soften the dermis by inhibiting the secretion of some stiffening factor(s) from the juxtaligamental cells in the dermis (Wilkie et al., 2004).
The viscoelastic nature of the dermis has been studied by carrying out various mechanical tests, and several mechanical models have been proposed (Motokawa, 1984b; Szulgit and Shadwick, 2000; Motokawa and Tsuchi, 2003). Among these studies, the one by Motokawa and Tsuchi (Motokawa and Tsuchi, 2003) is the most detailed. They carried out dynamic mechanical tests with a wide range of strain and strain rate on the isolated holothurian dermis. They concluded that the dermis assumed three different mechanical states that were distinguished not only by elastic properties such as elastic modulus but also by viscous properties and by strain-dependent behaviours. They referred to the dermis rested in ASW as in the standard state, that in KASW as in the stiff state, and that in Ca2+-free ASW as in the soft state. The mechanical parameters of the standard state did not show the simple intermediate value between those of the soft state and the stiff state, which led the authors to suggest that the stiffening mechanism altering the soft state to the standard state may be different from that altering the standard state to the stiff state. They also showed that the extent of the difference in the elastic modulus was maximal when measured under the dynamic strain imposed at the frequency of 0.3 Hz.
To understand the molecular mechanism of the stiffness changes in catch connective tissues, it is necessary to identify the cell-derived stiffening or softening factors that directly act on the extracellular matrix. Recently, a protein, tensilin, a candidate for the cell-derived stiffening factor, was isolated from the dermis of the dentrochirotid sea cucumber Cucumaria frondosa (Koob et al., 1999; Tipper et al., 2003). Tensilin retarded the speed of sagging of the dermis held horizontally at one end to allow downward bending by gravity in Ca2+-free ASW. This tissue-bending test gives no insight into standard mechanical parameters such as absolute stiffness and energy dissipation and thus we have no data to assess how much tensilin contributes to the extremely large changes in the mechanical properties of the dermis. A more quantitative mechanical test is necessary to understand the change in mechanical properties induced by tensilin. Moreover, we do not even know if tensilin has a role in the stiffness changes in vivo because tensilin has never been tested on dermis containing Ca2+. As the dermis, whose mineral content is in equilibrium with seawater (Koizumi, 1935; Trotter et al., 1997), contains ∼10 mmol l–1 Ca2+, we might better reserve nominating tensilin as a candidate for stiffness-controlling proteins in vivo until it is proven to work under a natural ionic condition. We also do not know whether tensilin is a general stiffening factor within the class Holothuroidea because no attempts have been made to test it on the dermis of different holothurian species or to isolate similar proteins from other species.
In the present paper, we isolated a homologue of tensilin from the dermis of the sea cucumber Holothuria leucospilota, which belongs to the Aspidochirotida, a different family to that of the sea cucumber from which the known tensilin was obtained. Dynamic mechanical tests were employed to quantitatively describe the extent of stiffness and energy dissipation changes. The effect of the homologue of tensilin (H-tensilin) was examined under normal ionic conditions with and without Ca2+. The present study showed that H-tensilin increased the stiffness in the soft dermis up to the stiffness level of the non-stimulated resting state (standard state), but it did not cause any further increase to reach the high level comparable to the stiffness found in the dermis in the stiff state.
Materials and methods
Animals and tissues
Specimens of the aspidochirotid sea cucumber Holothuria leucospilota Brandt were collected near Sesoko Marine Science Center, the University of the Ryukyus, Okinawa, Japan. They were shipped to Tokyo Institute of Technology and kept in an aquarium with closed circulating seawater at 20–23°C. The body wall is made of (starting from the outside) the pigmented epidermis, the thick dermis and the muscle layers, whose coelomic surface is covered by the coelomic epithelium. Unlike dendrochirotid sea cucumbers, the dermis has no apparent separation into outer and inner layers. We used the middle region in thickness of the dermis for both isolation of a stiffening factor and for mechanical tests. For mechanical testing, columnar test pieces were dissected out from the dorsal interambulacral region with a razor blade. Their long axis, along which tensile strain was applied, corresponded to the long axis of the animals. The size of the test pieces was 2×2×7 mm.
Dynamic mechanical test
Artificial seawater with normal composition (nASW) had the following composition: 0.50 mol l–1 NaCl, 50 mmol l–1 MgCl, 10 mmol l–1 KCl, 10 mmol l–1 CaCl2 and 10 mmol l–1 3-(N-morpholino)-2-hydroxypropanesulphonic acid (MOPS). In Ca2+-free ASW, the CaCl2 was replaced with 7.2 mmol l–1 ethyleneglycol bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). When the effect of elevated concentration of K+ was investigated, a high K+ stock solution (0.51 mol l–1 KCl, 50 mmol l–1 MgCl2, 10 mmol l–1 CaCl2 and 10 mmol l–1 MOPS) was added to the test solution to raise the KCl concentration to 100 mmol l–1. The pH of solutions used in mechanical tests was adjusted to pH 8.0.
Dynamic tests at the frequency of 0.3 Hz were performed on the isolated dermal pieces. As mentioned in the Introduction, the detailed mechanical dynamic tests covering a wide range of frequencies showed that the frequency of 0.3 Hz reveals the greatest differences between the stiffness value in the stiff state and that in the standard state (Motokawa and Tsuchi, 2003). The dermal pieces were subjected to sinusoidal tensile strain that ranged from 0 to 30% in a cycle. The stress at 0 strain was 0 at the beginning of the mechanical test. The details of the testing device have been described elsewhere (Motokawa and Tsuchi, 2003). As the maximal tensile stress occurred at the maximal strain in a cycle, the maximal stress divided by the maximal strain was defined as stiffness. The stiffness 500 s after the application of chemicals was compared with the value just before the application because the stiffness had reached a plateau or was close to a plateau 500 s after the application. The relative stiffness was calculated by dividing the value 500 s after the application by the value before the application. The mean values of stiffness were given in geometric means. The statistical differences between means of stiffness were tested by t-test on the log-transformed data. One cycle of deformation generated a hysteresis loop that was composed of a loading curve and an unloading curve. The energy dissipated corresponded to the area enclosed by the loop. It was expressed as a percentage of the deformation energy, which was the area under the loading curve (Motokawa and Tsuchi, 2003).
The dermis samples tested in Ca2+-free ASW were incubated in the same solution at 4°C for 1 h and then at room temperature for another hour before use. The dermis tested in nASW was rested for 20 min in a trough filled with the same solution.
The experimental trough contained 0.9 ml of the bathing solution, either nASW or Ca2+-free ASW, to which 0.1 ml of a test fraction was added. The test fraction was added after the stiffness value became steady (see Results). When the ionic composition of the fractions was different from that of the bathing solution, the fractions were dialyzed against the bathing solution before mechanical tests. The concentration of the purified stiffening factor used in mechanical tests was 1–3 μg ml–1. The stiffness values given in Results were for a concentration of 3 μg ml–1. The mechanical tests were performed at room temperature (20–27°C), which did not change more than 1°C during a test.
Purification of stiffening factor
The dermis was minced in two volumes of 2 mol l–1 NaCl, 10 mmol l–1 EGTA, 20 mmol l–1 Tris-HCl, pH 8.0. It was frozen at –80°C for at least 3 h and then thawed on ice. It was homogenized, frozen again and re-thawed, and was centrifuged at 27 000 g for 30 min at 4°C. The supernatant was used to purify the stiffening factor and the precipitate was used to isolate collagen fibrils (see below). The supernatant was precipitated with 60% saturated (NH4)2SO4 and the precipitate was then dissolved in 0.5 mol l–1 NaCl, 2 mmol l–1 EGTA, 20 mmol l–1 Tris-HCl, pH 8.0 (Tris-HCl buffer) and dialyzed extensively against the same solution. After centrifugation at 300 000 g for 30 min, the supernatant was applied to an anion exchange chromatography column (Mono-Q; Pharmacia Biotech, Piscataway, NJ, USA) that had been pre-equilibrated with the same buffer. The column was washed with the same buffer, and stepwise NaCl elutions were successively applied (0.65, 1.0 and 2.0 mol l–1) at a flow of 0.1 ml min–1. The fraction with strongest stiffening activity (0.65–1.0 mol l–1 NaCl fraction; see Results) was then loaded onto a gel filtration column (Superose 6 HR 10/30; Pharmacia Biotech) equilibrated with Tris-HCl buffer. Stiffening activities of fractions obtained in all the purification processes were assayed by dynamic mechanical tests on the dermis in Ca2+-free ASW. Protein concentrations were measured with a BCA protein assay reagent kit (Pierce, Rockford, IL, USA) using bovine serum albumin as a standard.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (Laemmli, 1970). The gels were stained with Coomassie Brilliant Blue R-250 to visualize proteins. To detect sugar chains, proteins in the gel were transferred (100 mA for 1.5 h) to polyvinylidine disulfide (PVDF) membrane, which was stained with a G. P. Sensor stain kit (Honen Corporation, Tokyo, Japan).
Partial amino acid sequences of stiffening factor
Purified stiffening factor was digested with trypsin (from bovine pancreas, TPCK-treated; Sigma, St Louis, MO, USA) and resulting fragments were applied to a reverse-phase high-performance liquid chromatography (HPLC) column (Inertsil 300C8; GL Science Inc., Tokyo, Japan) separated by application of a linear gradient of 10–60% acetonitrile in 0.1% trifluoroacetic acid. N-terminal amino acid sequences of undigested stiffening factor and a tryptic fragment were determined with a protein sequencer (PPSQ-10; Shimadzu, Kyoto, Japan). Obtained sequences were compared with a known sequence of tensilin from Cucumaria frondosa using the National Institute of Genetics website (www.nig.ac.jp).
Isolation of collagen fibrils and aggregation assay
The precipitate obtained from 10 g wet mass of dermis (see above) was suspended in 20 ml of 3 mol l–1 NaCl. The suspension was centrifuged at 27 000 g for 10 min at 4°C, and the precipitate was re-suspended in 3 mol l–1 NaCl. The cycle of precipitation and re-suspension was repeated four more times, and the precipitate was suspended in 20 ml of distilled water. The centrifugation and re-suspension in distilled water was repeated four more times and the precipitate was suspended in 40 ml of 3 mol l–1 guanidine-HCl. After the same centrifugation, the precipitate was suspended in Tris-HCl buffer. The centrifugation and re-suspension in the buffer was repeated three times to remove guanidine-HCl. Trypsin (1 μg ml–1) was added to the suspension, and the suspension was incubated for 12 h at 25°C. Centrifugation and resuspension in Tris-HCl buffer without trypsin was repeated three more times. We observed long fibres similar to reported collagen fibrils (Trotter et al., 1995) under a light microscope. The fibrils were re-suspended in the test solution used in the following aggregation assay.
Suspension (20 μl) that contained collagen fibrils from 1.4 mg dermis was mixed with the same volume of test solution either in the presence of calcium (10 mmol l–1 CaCl2, 0.5 mol l–1 NaCl, 20 mmol l–1, Tris-HCl, pH 8.0) or in its absence (2 mmol l–1 EGTA, 0.5 mol l–1 NaCl, 20 mmol l–1 Tris-HCl, pH 8.0). Whether or not the collagen fibrils were aggregated was observed by eye (see Trotter et al., 1996).
Purification and characterization of stiffening protein
The freeze–thaw extract showed slight stiffening activity. Separation on an anion exchange column yielded stiffening fractions eluted from the column with 1.0 mol l–1 NaCl (Fig. 1). They were further separated on a gel filtration column (Fig. 2), and a fraction was obtained with stiffening activity, which contained only a single major protein in SDS-PAGE. The apparent molecular mass from SDS-PAGE was 34 kDa, which is similar to the value of tensilin from Cucumaria frondosa. The apparent molecular mass of tensilin, a protein of 260 residues in the predicted mature form, is 33 kDa (Tipper et al., 2003). The yield of the purified stiffening protein from 1 g of dermis (wet mass) was 2.5μ g. The 34-kDa protein migrated at the same speed in SDS-PAGE regardless of the presence or absence of a reducing agent (Fig. 3A), suggesting that the protein is monomeric and contains little, if any, intrachain disulfide bonds. No sugar chain was detected by G.P. Sensor (Fig. 3B).
Partial amino acid sequences
The amino acid sequence of the N-terminus of the stiffening protein was Trp-Gly-Gln-X-Ser-X-Asn (X represents an unidentified amino acid). Among the tryptic fragments, we found one with the sequence Trp-Gly-Gln-His-Ser-Thr-Asn-His-Pro-Gln, which matched the N-terminus. Five of 10 amino acid residues of this sequence were identical to those of the N-terminal region of mature tensilin from Cucumaria frondosa (Tipper et al., 2003) (Fig. 4). Thus, we regarded the fragment as the N-terminus. Another tryptic fragment with 15 amino acid residues was sequenced; it had 33% homology to a known sequence of tensilin (Fig. 4). As discussed later, our data including the stiffening activity, molecular mass and amino acid sequences strongly suggested that the present protein from Holothuria leucospilota is a homologue of tensilin from Cucumaria frondosa. Thus, we called the stiffening protein from Holothuria leucospilota H-tensilin; the tensilin from Cucumaria frondosa is hereafter called C-tensilin.
Effect of H-tensilin on stiffness and dissipation ratio in Ca2+-free dermis
In all the dermis pieces in Ca2+-free ASW, stiffness decreased to about one third soon after the start of mechanical testing. Because the strain we used exceeded the maximum strain for induction of stress softening, this softening very likely corresponded to the stress softening characteristic of the dermis in the soft state (Motokawa and Tsuchi, 2003). The stiffness became constant or the rate of decrease became small in a few minutes. The stiffness 5 min after the onset of mechanical testing was 1.3 kPa (Table 1). The application of H-tensilin increased the stiffness whereas the addition of the same amount of Ca2+-free ASW without H-tensilin did not change its stiffness (Fig. 5). The relative stiffness increased by 3–15-fold. The effect was apparent in a few minutes and the stiffness reached a plateau in 4–10 min in most cases. After reaching a plateau, the stiffness changed little for up to 30 min. When H-tensilin was removed by washing with Ca2+-free ASW, stiffness decreased to the level before the application of H-tensilin (Fig. 5B). The mean stiffness in H-tensilin was 8.9 kPa, which was statistically different from the value before application of H-tensilin (P<0.001, N=6) by t-test.
A single deformation cycle generated a hysteresis loop with a loading curve and an unloading curve. In Ca2+-free ASW before the application of H-tensilin, the unloading curve did not follow the preceding loading curve; the stress in the unloading curve was much lower than that of the loading curve (Fig. 6A). Therefore, the dissipation ratio was as high as 52.3% (Table 2). The addition of H-tensilin made the difference between the two curves smaller, and thus the dissipation ratio became smaller (Fig. 6B). The mean dissipation ratio was 33.3%, which was statistically different from that before application of H-tensilin by paired t-test (P<0.05, N=6) (Table 2).
Effect of H-tensilin on dermis in nASW
The dermis in ASW did not show stress softening at the onset of mechanical testing except in one sample. Other samples showed a slight decrease in stiffness or a transient increase in stiffness by ∼2-fold for the first 10–15 min and then the stiffness became rather constant (Fig. 7). The mean stiffness in nASW was 16.6 kPa, which was statistically different from that in Ca2+-free ASW without H-tensilin (P<0.001) by t-test (Table 1) but was not different from that in Ca2+-free ASW with H-tensilin. The mean stiffness was greater than the highest stiffness value (13.5 kPa) of the H-tensilin-treated dermis in Ca2+-free ASW. Quite a large variation, ranging from 4.5 kPa to 98.7 kPa, was observed in nASW, as was reported previously (Hayashi and Motokawa, 1986; Motokawa, 1984c). The application of H-tensilin had no effect in eight out of nine samples tested (Fig. 7B). The mean stiffness after the application of H-tensilin did not differ from 1 before the application (Table 1). In one sample, H-tensilin increased the relative stiffness by 1.39-fold. This sample was exceptional both in responding to H-tensilin and in showing stress softening at the start of mechanical testing. The stiffness of this sample before the application of H-tensilin was 4.57 kPa, which was closest to the lowest value of 4.52 kPa in nASW.
The dissipation ratio of the dermis in nASW before application of H-tensilin was 38.6% (Table 2). The application of H-tensilin did not change the shape of the hysteresis curve and thus did not change the dissipation ratio: the mean ratio was 37.6%, which was not statistically different from the value before application of H-tensilin (Table 2). The dissipation ratios in nASW, with or without H-tensilin, were not statistically different from the ratio in Ca2+-free ASW with H-tensilin.
Artificial seawater with the potassium concentration raised to 100 mmol l–1 was applied. This treatment caused a marked stiffness increase in the dermis that did not respond to H-tensilin (Fig. 7B). The stiffness reached a plateau about 500 s after the elevation of the concentration of K+. A similar increase in stiffness in response to 100 mmol l–1 K+ was also observed in the control dermis (Fig. 7A). The mean stiffness in the high K+ solution was 123.0 kPa, which was statistically different from those in nASW with or without H-tensilin (P<0.05) by t-test and from that in Ca2+-free ASW with H-tensilin (P<0.01) (Table 1). The mean dissipation ratio in the high K+ solution decreased to 19.3%, which was different from means in nASW with H-tensilin and without H-tensilin by t-test (P<0.05) (Table 2).
Collagen fibril aggregation by H-tensilin
A clot of fibrils was formed in the collagen-suspension solution when it was mixed with an equal amount of buffer solution containing H-tensilin, whose concentration was 20 μg ml–1 after mixing. The fibril formation was observed both in media with Ca2+ and without Ca2+ (Fig. 8).
H-tensilin is a homologue of C-tensilin
We have isolated from the sea cucumber Holothuria leucospilota a protein that stiffened the dermis in Ca2+-free ASW. The protein induced aggregation of collagen fibrils in vitro. The protein seems to be a single chain because it behaved as a single band in SDS-PAGE with a reducing agent. It contained no detectable sugar chains. These characters, together with the apparent molecular mass and amino acid sequences, strongly suggested that the protein was a homologue of tensilin (C-tensilin) isolated from the sea cucumber Cucumaria frondosa (Koob et al., 1999; Tipper et al., 2003) and thus we called the present protein H-tensilin. Our finding that tensilin exists in two different holothurian families strongly suggests the wide distribution of tensilin in sea cucumbers. Although H-tensilin was similar in many respects to C-tensilin, they have a few different characters including the amino acid sequence. H-tensilin eluted from an anion exchanger at NaCl concentrations between 0.65 and 1.0 mol l–1 at pH 8.0 whereas C-tensilin eluted from anion exchange chromatography between 0.2 and 0.3 mol l–1 NaCl (Koob et al., 1999; Tipper et al., 2003). This difference suggests that H-tensilin is more negatively charged than C-tensilin at pH 8.0. Another difference was observed: H-tensilin had only a small number of intrachain disulfide bonds, if any, whereas C-tensilin had them in significant numbers judging from the migration speed in SDS-PAGE with or without a reducing agent (Tipper et al., 2003).
H-tensilin stiffened the dermis in the soft state
Motokawa and Tsuchi, based on their detailed mechanical tests in the holothurian dermis (Motokawa and Tsuchi, 2003), distinguished three different mechanical states: the soft state, the standard state (intermediate state) and the stiff state. The soft state could be induced in isolated dermis by immersing it in Ca2+-free ASW (Motokawa and Hayashi, 1987); most of the isolated dermal pieces in nASW were in the standard state; the stiff state could be induced in isolated dermis by immersion in ASW with elevated K+ concentration. The soft state was distinguished from the standard state by low stiffness, a high dissipation ratio and stress softening. The present study confirmed these features. In Ca2+-free ASW, every dermis showed stress softening at the start of mechanical testing and the stiffness was one-seventh of that in nASW. H-tensilin had a stiffening effect on the dermis in Ca2+-free ASW, an effect that was already known for C-tensilin. The quantitative mechanical tests revealed that H-tensilin increased the dermal stiffness to the same level as that of the dermis in nASW and that H-tensilin decreased the dissipation ratio to the same level as that of the dermis in nASW. Therefore, H-tensilin converted the dermis in the soft state to the standard state.
The dermis is known to have a similar ionic environment to nASW (Trotter et al., 1997). The result that H-tensilin had no effect in nASW might infer that tensilin was not responsible for the stiffness changes in vivo. It should be noted, however, that Ca2+ depletion was not the essential condition for producing the soft state. The isolated dermis in nASW softened in response to various stimuli such as acetylcholine, holokinin, an endogenous peptide of sea cucumbers, and very strong mechanical stimulation (Motokawa, 1987; Motokawa, 1988; Birenheide et al., 1998), and thus the dermis could take the soft state with the presence of normal Ca2+ concentration in bathing media. The exceptional case in nASW of the present study did show that tensilin could exert effects, although not strong, under the normal ionic condition with Ca2+. This exceptional sample in nASW was not only exceptional in responding to H-tensilin: it was the only sample that showed stress softening and was among the two dermis samples with exceptionally low stiffness in nASW. The stress softening and the low stiffness strongly suggested that it was in the soft state. The possibility has been discussed that, among the isolated dermis in nASW, some were in the soft state and some were in the stiff state, judging from the extremely wide range in the parameters of mechanical properties of the isolated dermis in nASW (Motokawa, 1984c). The wide range in stiffness was also observed in the present study. Thus, we concluded that tensilin had a stiffening effect on the dermis in the soft state irrespective of the presence or absence of Ca2+.
H-tensilin did not produce the stiffest dermis
Based on the levels of stiffness and dissipation ratio and the absence of stress softening, we regarded the dermis in nASW as being in the standard state, with the exception of the one unusual sample discussed above. Instead of stress softening, transient stiffening was observed in nASW several minutes after the onset of dynamic tests in half of the samples. Similar stiffening was reported in other sea cucumbers in nASW (Shibayama et al., 1994). The dermis very likely took the cyclic stretching as mechanical stimulation and responded by stiffening (Motokawa, 1984a). H-tensilin had no effects on the dermis in the standard state; subsequent application of ASW with elevated K+ concentration increased the stiffness by one order of magnitude. Therefore, H-tensilin could not account for the stiffness changes from the standard state to the stiff state. The result that H-tensilin did not change the dermis in the standard state to the stiff state strongly suggested that some factor(s) other than tensilin was needed to produce the stiffest dermis. The ASW with elevated K+ was known to induce the stiff state through stimulating nervous or other cellular elements (Motokawa, 1994). The search for a cell-derived factor(s) other than tensilin looks promising; indeed, we have already isolated, from the present sea cucumber species, the fraction that had stiffening effects on the dermis in nASW (A.Y., M.T., K.O. and T.M., unpublished).
The present study showed that H-tensilin converted the dermis in the soft state to the standard state but it did not convert the dermis in the standard state to the stiff state. This finding, in turn, could be taken as the chemical evidence for the presence of the three different states that have been inferred through the mechanical studies (Motokawa and Tsuchi, 2003).
Molecular mechanism of stiffening by tensilin
The mechanical behaviour of the dermis in the soft state suggested the presence of two kinds of bonds between force-bearing molecules. The stress softening suggested the presence of bonds irreversibly broken by a large strain. After the stress-softening has completed, the stress–strain curves remained constant: in each cycle, the loading curve always showed higher stiffness than the unloading curve at the same strain. This result suggested the presence of bonds that were temporarily broken at stretching and were recovered when the tensile strain was removed (Motokawa and Tsuchi, 2003). The dermis in the standard state neither showed the stress softening nor the difference in the loading and unloading curves. This suggested that the bonds not broken by strain were dominant in the standard state. Either new bonds were introduced or the labile bonds were converted into nonlabile ones at the transition from the soft state to the standard state. We still do not know how tensilin exerted effects on these changes. Ca2+-free ASW is believed to decrease stiffness by inhibiting the secretion of tensilin from juxtaligamental cells and thus decreasing the extracellular concentration of tensilin in the dermis (Wilkie et al., 2004). The ability to induce collagen-fibril aggregation in vitro, which was shown for H-tensilin (present study) and for C-tensilin (Tipper et al., 2003), might suggest that the aggregation and disaggregation of fibrils directly corresponded to changes in the bonds between force-bearing molecules. We should be cautious, however, as there is a report of a dermal protein, stiparin, with no stiffening effects on the holothurian dermis causing collagen-fibril aggregation (Koob et al., 1999).
Tipper et al. reported that the peptide sequence of C-tensilin had 30% identity to tissue inhibitor of metalloproteinases (TIMP) (Tipper et al., 2003). A sequence (His-Pro-Gln) common to TIMP (Montagnani et al., 2001) was present in C-tensilin. H-tensilin also had this sequence in the probable N-terminal region. The stiffening activity of tensilin might be through the TIMP activity, but this has not been demonstrated yet. The present study, however, did not support this possibility. H-tensilin lacked two cysteine residues near its N-terminus, which were present both in TIMP and in C-tensilin (Montagnani et al., 2001; Tipper et al., 2003). The lack in H-tensilin was consistent with the result of SDS-PAGE showing little presence of disulfide bonds. In human TIMP-1, these cysteine residues near the N-terminus form intrachain disulfide bonds (Cys1–Cys70 and Cys3–Cys99) (Williamson et al., 1990) that were suggested to be important for TIMP activity (Gomis-Rüth et al., 1997). The location of cysteine residues in other TIMPs is conserved, and thus the disulfide bonds are probably conserved to play important roles in TIMP activities (Montagnani et al., 2001). The lack of such bonds in H-tensilin does not favour the idea that TIMP activities are involved in the rapid changes in dermal mechanical properties.
We thank the staff of Sesoko Marine Science Center, the University of the Ryukyus for providing specimens. Supported by Grant-in-aid for scientific research on priority area (A) `Molecular synchronization for design of new materials system' of the Ministry of Education, Science, Sports, and Culture of Japan to T.M.
- © The Company of Biologists Limited 2006


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by Krystal Brown
Making your own soap is an enjoyable and useful activity. Homemade soaps are also better for the skin because they contain glycerin. Glycerin is a natural product of the soap making process. But, with many commercial soaps, the glycerin is removed and used to make lotions and other cosmetics.
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When starting out, it is better to keep things simple and low cost. I am going to teach you how to make an uncomplicated soap using just four ingredients.
Those ingredients include Crisco, orange essential oil (which is optional), lye and olive oil. You don't have to use any essential oils if you want a fragrance free soap. Usually making soap from scratch involves a "cure" time of 4-6 weeks. During this time, the soap becomes milder and harder.
However, I am going to also teach you a simple oven processing technique that can make your soap safe to use within a couple of days.
Items Needed
- Goggles: This is to protect your eyes from the "fumes" generated by the lye water. I have used my old swimming goggles and they worked just fine. You can also get goggles from the Dollar Tree.
- White Vinegar: This is used to neutralize the lye if any of lye water splashes on you.
- Rubber Gloves: These protect your hands and can also be obtained from most dollar stores.
- Stick Blender: I purchased mine for less than $10 from a local discount store.
- Silicone Cake Molds: I got some from Dollar Tree. I used 5 small silicone cake molds for my soaps. You can also use a regular pan lined with lightly oiled wax paper so that the soap will not stick to the pan.
- Candy Thermometer: The first time I ever made soap, I didn't have one of these and it made things harder. Get a candy thermometer. I purchased one for a couple of dollars on eBay.
- Lye: I use "Instant Power Crystal Lye Drain Opener" from Walmart. It costs $5.16 and I still have plenty of lye left over to make more batches of soap. Your lye must be 100% pure lye…not Drano or anything like that. You can also purchase lye from eBay, Amazon or various online soap making shops. I have heard of people buying the 100% pure lye from Lowe's as well. You will need ¾ cup of lye for this recipe.
- Crisco: You will need 3 pounds of this. I got 3 containers of Crisco from Dollar General. Each container was a pound. You can use generic Crisco as well but it must be 100% vegetable shortening.
- Olive Oil: You will need 1 tablespoon of this. I purchased mine from Dollar General. It must be 100% pure olive oil, not the "olive oil" blend sold at Dollar Tree which is mainly just soybean oil. The olive oil hardens the soap.
- Orange Essential Oil: I used about ½ of an ounce. It smells heavenly. You can get essential oils on eBay, Amazon and at drugstores such as Rite Aid.
- Kitchen Scale: This is optional for this recipe (in my opinion). It will make things more precise, but I did not use a scale because I did not have one at the time. I just measured things out carefully using measuring cups and I had no problems.
The 12 Easy Steps
- Measure out your lye. If you have a scale, measure out 220 grams of lye. If not, very very carefully measure out ¾ cup of lye and pour the crystals into a non-metal container.
- Measure out 330 grams (or 1 and ½ cup) of distilled or filtered water. Very carefully pour the lye crystals into the water. There will be fumes. Stir with a spoon (again no metals are allowed…use plastic or wooden spoons). It is good to place the lye water under the stove exhaust fan if you can. If not, open the windows for adequate ventilation.
- Melt your Crisco and add your olive oil.
- After the oils are melted together, turn off the stove and remove the pot from the stove. Use a thermometer to measure the temperature of the oils. The oils should be about 120 degrees.
- Check the temperature of your lye water. It will be about 175 degrees.
- You must get the lye water and the oils to a similar temperature. So, place your lye water container in a sink full of ice water until it has cooled down to 97-100 degrees. While you are waiting for this to happen, your oils will be cooling down naturally.
- When both the oils and the lye water are between 97-100 degrees, mix them together. Pour the lye water into the oils. Use your stick blender to blend the mixture until it looks like pudding. When the mixture reaches this stage, it is said to be at trace.
- When your mixture reaches trace, add your essential oils to the mixture. Blend well and quickly pour your mixture into your cake pans. Certain essential oils will sometimes cause the mixture to "seize" so work quickly once you add your fragrance oil.
- Place your soap in the oven at 170 degrees (or on the lowest setting your oven has available. If your oven has a "warming" feature, you can use that). Let the soap stay in the oven for about 2 hours. However, check on the soap every 20 minutes or so to make sure that it does not "bubble over". The heat from the oven is forcing the saponification process. If you didn't use the oven method, it would take 4-6 weeks after you pour your soaps before you could be able to use them.
- Let your soap rest for a few hours and then cut it into soap bars.
- Let the soap rest for another day so the excess water will evaporate and the soap will become milder and harder.
- Enjoy your homemade soap!
This bar cleans very well and has a medium amount of lather. Many people (myself included) love lots of lather. But, the amount of lather a soap produces does not measure its cleaning ability. My Orange Crisco Soap is not extremely moisturizing like a Dove beauty bar, but it does not strip the skin either.
Your entire family can use it or you can even sell the soap at flea markets or online. The ingredients are cheap and readily available which make it perfect for a novice soap maker. I showered with my soap 2 days after making it. I have a whole box of soap from this one batch. It's easy, economical and fun. Please try it!
Also Read:
- DIY Rendering Fat & Making Soap
- Judy's Homemade Laundry Soap
- Soap Thieves, A Surprising Lesson About Prepping
- Making and selling soap
- Making Laundry Soap
- Making Soap From Scratch: How to Make Handmade Soap – A Beginners Guide and Beyond
M.D. Creekmore
Latest posts by M.D. Creekmore (see all)
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subset: commoncrawl


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text: The Dips always loved those melodramatic operatic chants—most ridiculous on Juelz's "Monster Music"—but they're used here to menacing effect. Even though Killa Season failed to live up to the lofty standards set by its predecessor, Purple Haze, this is one of the album's few records that carried the same genetic material. Sort of like when Patrick Ewing Jr. made a couple 14-foot jumpers before getting cut in preseason.


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Steve Schmidt, McCain Campaign Manager: Religion Could Kill The GOP
Mr. Schmidt made these comments in a speech to the Log Cabin Republicans, a gay group trying to affect the Republican party from within. (Like Newt, he has a gay sister.) In other words, the L.Cs is a group that is heavily into denial! It remains to be seen if the Repubs can ever divorce themselves from their ties with religion or their anti-gay bias but so far, either option seems far from the GOP's agenda.
Right now, their activity du jour has to do with Tea Parties and "teabagging" (which I'm sure they are expert at!) and I'm sure they are trying to protect the "poor" (themselves) from high taxes! They've had a free ride for 8 years and now they object. More "patriotism?
I'm also not sure why the Obamas want to pardon all those employees at Gitmo who were just "following orders" in the really "benign" (no torture!) interrogation of terror suspects. In doing this, Barack is putting himself in the same category as these other creeps. And, I might add, I don't understand the consistent appointments oƒ those who are supposed to get us out of the present "recession" with those who got us into it. (Geithner, Summers, Allison.) So far, they've only succeeded in throwing away more taxpayer dollars.
Though I don't regret voting for Obama (would McCain have been any better?), I have been rather disappointed in many of his subsequent actions.
About GOP
Read the Article at HuffingtonPost


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Somerset Beekeepers in the news
Did you see a group of Somerset beekeepers in The Times and the Guardian earlier in the month? Photographer Zach Culpin went along to an apiary session for Somerton's beginners' group and took a series of colourful photos which he kindly shared with us. Great work Joe King, Stewart Gould and the new beeks!
SW Regional Bee Inspector Simon Jones writes about the last season in his annual report - the good, the bad and the ugly! It provides a fascinating snap-shot of what has happened in Somerset and the region.
There is some good news for our county beekeepers - the number of colonies affected by EFB fell last year to 26, but that is still more than Devon, Cornwall and Avon put together. However AFB was not reported in Somerset but it is just over the county border in Devon.
"The 2018 season started late after a cold winter and spring. However colonies that were healthy and strong coming out of the winter were able to take advantage of the good weather that followed and gathered a good crop of honey. From my own experience the honey has been of a good quality with most of the combs fully capped with the extracted honey having a low moisture content.
The Bee Inspectors have had another busy season. They have been carrying out their usual programme of risk based inspections for European Foulbrood (EFB) and American Foulbrood (AFB). There has been an increase in the levels of disease in Cornwall but the other counties which make up the South West Region (SW) have showed a significant decrease with Avon having no disease at all (see graphs further in the report).
In partnership with the local Beekeeping Associations we have continued to deliver our 'Bee Health Days'. This season the North Somerset/Avon Bee Health Day took place at the Langford Veterinary College, the Cornwall Bee Health Day was in Lostwithiel at the local community centre and the Devon Bee Health Day was hosted by Exeter Branch at the Kenn Centre just outside of Exeter.
I would like to thank all of the local association secretaries/training officers who helped us to manage the programme of Bee Health Days across the SW. We are also very grateful to the local beekeepers who allowed us to use their nearby apiaries for demonstration purposes.
The Inspectors also had a presence at the Somerset Lecture Day in February and Cornwall's 'Bit of a Do' (BOAD) in September.
We have again been carrying out Exotic Pest Surveillance (EPS) inspections around risk points such as ports and airports to check for incursions of Small Hive Beetle (SHB), Tropilaelaps and Asian Hornet. With local beekeepers help we set up four Enhanced Sentinel Apiaries, two are situated near Avonmouth Docks and two near Plymouth Docks, both are considered to be very high risk points. These apiaries are inspected three times a year by the local BeeInspector and floor samples are sent into the FERA laboratory for analysis.
In addition to these inspections there are 18 Sentinel Apiaries around the region. This is where beekeepers that have an apiary near a risk point assist the National Bee Unit (NBU) by regularly monitoring their colonies for Exotic Pests and twice a year they send in floor debris samples to be tested. I am very grateful to the South West Sentinel Apiary Beekeepers for all of their help this season, they are an important part of the NBU's surveillance programme.
Part of our work each season is to monitor imports of queens and package bees from the European Union (EU) and Third Countries (Argentina, Australia and New Zealand). Again this year we saw a large number of packages imported from Italy, some of which were bought by beekeepers in the South West (SW) region. These were inspected for SHB as they were decanted into hives and fortunately nothing was found (SHB was found in Southern Italy in 2014).
Throughout the season the SW Inspectors carried out enhanced surveillance for Asian Hornet in the Woolacombe area of North Devon to ensure nothing had been overlooked from the nest that we found and destroyed in 2017.
We were also in the thick of it again when Asian Hornet was found to be predating bees in an apiary in Cornwall."
For more a detailed report, please download the file below.
All members of SBKA are to be given an Asian hornet trap and the attractant Suterra.
We anticipate starting to distribute the Veto Pharma traps at Lecture Day on February 16 in good time before any over-wintering queens start to emerge.
The remainder of the traps and the attractant will be available for members to collect from their Divisions.
We are determined to stop Asian hornets becoming established in the county and to that end AHATs (Asian Hornet Action Teams) have been set up, publicity materials produced and a PR-campaign started.
Last November Council set a £6,000 budget for our 2019 Asian hornet campaign and we subsequently received a £3,600 donation from Yeo Valley which Council decided to allocate to this work.
The costs of providing the traps and attractant will be £4,200; £3,400 being the cost of the traps supplied by Thornes at a slight discount and £800 for Suterra bought in bulk.
Members will receive Asian hornet fliers and identification cards, plus an EFB leaflet, with their 2019 year books. Pick up your year book at Lecture Day; the remainder will be posted.
As soon as day time temperatures reach 10-12 degrees C, any over-wintering queens will emerge. Time for super-vigilance to start!
Beekeeping in the snow!
It's worth checking your hives today and clearing the snow from landing boards to ensure entrances don't get blocked up with snow or ice if there's a thaw followed by freezing temperatures. And don't worry if you see the odd dead bee which has been attracted by this morning's bright light and then succumbed to the cold.
Report of BBKA ADM 2019
Richard Bache is our BBKA Delegate and reports from the recent BBKA ADM.
On the 12th January I attended Myton School in Warwick for the 59th BBKA ADM. A minute silence was held for the beekeepers that had passed away. There were the usual housekeeping, apologies and adoption of the standing orders. Margaret Murdin (President and Chairing the meeting) reported that there had been increasing abuse over the telephone by beekeepers towards the staff at BBKA headquarters necessitating the introduction of telephone recording. 66 (of 74) Area Associations were present, representing 23284 members.
Minutes and Reports
An amendment from Manchester regarding the minutes of the 2018 ADM had been accepted by the executive. Bob Maurer (Surrey) asked why there was so little recorded about the disciplinary process at last years ADM. Margaret Murdin explained that it was best to leave it out as a lot of what was said was contentious and confidential. The minutes were accepted.
Terry Hitchman (Stratford delegate) asked what progress had been made on the discouragement of honeybee importation. Margaret Murdin stated that lobbying had occurred and that banning importation of honeybees might be possible after Brexit, but there were other political priorities for the government. It was noted that it was mainly bee farmers who were importing honeybees.
Tony Lindsell (Devon) recorded his disappointment with progress reports and the fact that it did not seem that ADM propositions moved freely to the EC to action. He also noted missed opportunities regarding a DEFRA consultation, although Anne Rowberry noted that she had responded in several capacities, including as BBKA trustee. Later, Tony Lindsell asked for a copy of the BBKA response to the DEFRA consultation.
John Canning (Cleveland) asked about the Northern Ireland situation post Brexit. Margaret Murdin noted that there were already difficulties in recording bees crossing the Northern Ireland border. The Northern Ireland (INIB) delegate stated that figures for bees crossing the border were not clear, there are bees and beekeepers that straddle the border and there are likely to be low numbers but also under-reporting.
Tony Lindsell briefly asked for an update on the VAT proposition of several years previously, although it was noted that this might be difficult to achieve before Brexit.
Roger Patterson noted that importation of honeybees was permitted from Argentina and that the have Africanised honeybees.
The reports were dealt with much quicker than in previous years as there were no verbal supplementary reports except on the Spring convention, where a paragraph had been omitted. It was explained that there was much subsidy of the convention by the traders and that they were trialling cheaper day tickets. Michel Badger (Yorkshire) asked whether there was a plan to stay with Harper Adams long-term. Joyce Nisbet replied stating that the venue was frequently under review, and although there were some disadvantages to the venue there were also many advantages and there are no plans to change the venue at present.
Simon Cavill explained that the next International Meeting of Young Beekeepers will be in Slovakia and that it was hoped to get beekeepers aged 12-16 involved for selection. The reports were nearly unanimously accepted.
Val Francis (for the exam board) noted that there had been more basic assessments and awarded the Surrey shield (Basic Assessments) to Newcastle and the George Knights Memorial Shield (higher assessments) to Gloucester.
It was noted that there had been a third consecutive year of surplus and no increase in capitation was intended. The reappointment of auditors and financial reports were both passed with near unanimous support.
New Association applications
After lunch, attention turned to the associations applying for BBKA membership. The Laddingford representative gave a fairly bitter account of their split with Kent BKA which seems to have centred around the insistence that all Kent members subscribe to Bee Craft. They were initially formed from the Yalding Branch and it sounds like there was deeper bitterness around the finances of the division. The Kent delegate offered no objections to them joining BBKA, but noted that some issues with their formation had been raised with the Charity Commission. The Medway representative also had no objections and supported the application. Bob Maurer (Surrey) made the point that once again, inability to sort out local issues had resulted in potential for increased administration at a national level. Michael Badger (Yorkshire) stated that they should pay an appropriate rate. As a delegate, the test I always apply is whether a new association represents fragmentation of existing associations or inclusion of previously unrepresented beekeepers. I felt this leaned towards fragmentation and therefore did not support the application. 34 Associations supported their application against 20 against, but this was overturned with a membership vote (7520 for, 10953 against).
Terry Hitchman (Stratford) reiterated a previous request that local problems could be sorted out by a national taskforce. David Coates (Burton and district, Derbyshire) stated that large associations wield too much power with the membership vote.
The other applicant association, Ellan Vannin, had withdrawn their application. Roger Patterson expressed annoyance that work had been undermined. Points were raised about the BBKA neutrality in membership applications before Michael Badger (Yorkshire) proposed we move to next business.
The executive nominees, John Canning, Margaret Wilson and Diane Drinkwater were duly elected as there were more candidates than vacancies. The Exam board were elected as Somerset wished with Stuart Roberts, Marin Anastasov and our own Lynne Ingram getting the three-year terms, Celia Perry getting the two-year term and Michael Cullen getting the one-year term.
After Lunch there was a rearrangement of the agenda to enable some work on the constitution.
Margaret Murdin explained that the VAT issue (from Devon) was best brought up after Brexit, and presented the past presidents badge to John Hendrie. There was a series of quick-fire non-contentious propositions which were heavily supported (including by me): Enabling videoconferencing (8), Winding up BBKA Enterprises (7) and changing the BBKA news wrapper to a recyclable material (5).
The proposition for protected time for propositions at the ADM (9) was felt to be too restrictive. Suggestions that were raised to enable more discussion included an informal forum, a digital forum or even a second day of voting. I was among 39 that voted against this one.
The first proposition to generate much debate was the inclusion of a schools category, should the new constitution not pass. It was clarified that one school would constitute one voting member and that the school would rely on their own insurance. It was agreed that the educational establishment would have to be defined more precisely with Ofsted registration (or Estyn in Wales). Annette Campbell (Newcastle) was concerned that all the students at the local university could converge on the club apiary, but Margaret Murdin stated that it was up to local associations to define the relationship they have with Educational establishments. Lesley Jacques (Cheshire) suggested that universities should be excluded as they were comprised of adults that could join independently. There was suggestion that the fact the wording was not published in time would make the proposition out of order, others suggested that it ought to go to the executive to work on it in more detail. However, two amendments were tabled- one to necessitate OFSTED/ESTYN registration for a school being a members (which I supported and passed) and one which included Universities (which I supported and did not pass). The proposition was passed (which I supported) with 56 votes (84%) for and 8 against. It was noted that it is up to associations to amend County membership classes accordingly.
The proposition on whether BBKA should be represented at tradex (6) divided opinion with many seeing an opportunity to engage with more beekeepers. It was noteworthy that those involved in the BBKA spring convention and those who were executive members when Bee Tradex was formed were very much against this, citing the inflexibility of the proposition. It was supported. The next proposition on publishing BBKA minutes divided opinion. Tony Lindsell (Devon) stated that this was a matter of transparency while Margaret Murdin felt that draft minutes could not be published and John Canning stated that it can suppress ideas at the meeting with decisions taken away from formal committee process. He instead pressed for a good report of the meeting. Margaret Wilson highlighted the positive thinking newsletter. It was suggested that the whole proposition be re-written. Padraig Floyd (Essex) proposed that the word Draft be omitted and I seconded this as this was the steer that I had from Somerset and this was well supported. John Canning then immediately proposed an amendment that this should be a summary of the meeting, which I felt watered down the proposition but was nonetheless passed and the amended proposition passed. The proposition to discourage manufacturers from selling leather gloves to beginners (11) was next, with the Derbyshire delegate stating that the best use for leather gloves is smoker fuel! Mike Duffin explained that he gets bad reactions and wears disposable gloves over the leather gloves. Anne Rowberry added that the suppliers had their reasons, but all agreed to suggest nitrile gloves. I was one of 44 who voted for this proposition.
The next proposition to be discussed was the discipline and dispute resolution policy (1). Stephen Barnes, for the executive, acknowledged that there were some weaknesses that had been identified. The broad change was that the right of appeal moved from being at the BBKA ADM to being independent arbiters. This, of course, would avoid the farcical events of last year where the EC were left potentially legally liable for the outcome of a dispute but had no input into the decisive vote. Padraig Floyd (Essex) supported the broad policy direction and vowed to work with the trustees to bring amendments to the next ADM. There were concerns that on one hand the BBKA needed a policy, but on the other, it needed to be well thought-out. The policy was accepted and collaboration with the review was invited.
BBKA Constitution
The major proposition was dealt with last: that of the new constitution (now that BBKA are becoming a charitable incorporated organisation). Stephen Barnes discussed the background to the document and the progress that had been made over the past year. He stated that there had been little feedback, but that it had been of high quality, and sometimes quite novel. There had been a number of amendments, many of which had been received in the previous 24 hours, ranging from minor to substantial. It was even considered whether to pull the document, but that they had identified 7 amendments for consideration:
The amended constitution was put to the vote and I supported it along with 58 other associations. The Manchester delegate expressed his thanks for all the effort that had been put into the document to widespread applause. Margaret Murdin thanked the delegates for their time and goodwill, and also the staff of BBKA.
AsIan Hornets - January update
As 2019 begins, our minds turn to the forthcoming beekeeping season, writes SBKA Asian Hornet Action Team Co-ordinator Lynne Ingram MB in the January issue of the newsletter.
At the moment we may be giving fondant to our bees, doing mid winter varroa treatments or perhaps planning our beekeeping goals for the year ahead. But, whatever else you are doing, you do need to be thinking about Asian Hornets.
Last year there were four nests found and destroyed in the UK, and nine confirmed sightings. As the numbers build up in Europe, and Northern France, the threat of Asian Hornets invading the UK increases, and we all need to be very vigilant in order to prevent/slow a major invasion. Asian hornets (Vespa velutina nigrithroax) are voracious predators of pollinators, and in particular honeybees, whose colonies contain a vast number of insects in one place – a veritable insect supermarket for the Asian Hornet!
So what are Asian Hornets doing this time of year?
Mated queens are now overwintering in cracks, crevices and sometimes underground. They can hibernate singly or in small groups. We don't know as yet, if there are any hibernating in the UK. Like bees they feed well in the Autumn, in order to build up their fat bodies to sustain them during the long winter. Like other members of the wasp family, they overwinter with their wings, legs and antennae protectively tucked in underneath them. The rest of the colony would have died out in December.
As the days lengthen in March or April (depending on the weather), the queens start to come out of hibernation, and firstly search for nectar rich flowers, in order to build up their energy. In France and Jersey they are often seen on early Camellia flowers, so if you have any, it may be worth keeping an eye out for Asian Hornet queens. Queens search for a nest site, then start creating golf ball sized embryo nests, which are generally in sheltered spots such as sheds or garages. To start with the queen will be laying the eggs, foraging for food and feeding the larvae. She may curl herself around the pedicel of the nest to keep the larvae warm. Once the first workers emerge, they will take over many of these tasks, leaving the queen to lay eggs. If you see a nest, check for Asian hornet activity, then photograph it before reporting it.
This is the time of year when a monitoring trap can be invaluable, attracting the queens to a sugar rich bait. It is important though that beneficial insects, such as European hornets and wasp queens, can be released from these traps in order to protect our native pollinators. You can make a trap from a water bottle as illustrated below (instructions on Beebase) www.nationalbeeunit.com/downloadDocument.cfm?id=1056
or buy a commercially available trap, and instead of filling the bottom with liquid bait in which insects would drown, scrunch up some kitchen towel so that insects can suck the bait from the paper without drowning. Or place a yogurt pot full of bait inside the trap, with a mesh or net covering to prevent drowning. Don't forget to inspect your traps regularly, and release anything else that has been captured.
If you find an Asian Hornet
If you do find an Asian hornet in your trap, the key is to get evidence so that you can report it. You could take a photo through the trap wall, or put the trap in a bag in the freezer, and then photograph the hornet once it is dead. Email your photo with details to: email@example.com or use the Asian Hornet Watch app.
Somerset Asian Hornet Action team
If you think you may have seen an Asian Hornet and are struggling to get evidence, or need some advice then contact the Somerset Action team on: firstname.lastname@example.com and someone will come and help with identification and getting evidence.
Go green for 2019!
The start of the New Year is an excellent time to look ahead to the coming season and plan what you'd like to achieve with your bees. Whether that's trying a new type of hive, creating more colonies or breeding your own queens (and this is the year to mark them green), then give it a go!
SBKA is focusing on helping all members recognise EFB through leaflets, new digital content and 'Read the Comb Day' on June 15.
Also, we want to alert everyone in Somerset (and beyond) to be on high alert for Asian hornets. We'll be running a publicity campaign via the press and our own social channels as well as working with organic, national dairy Yeo Valley to spread the message far and wide.
If you have connections with local gardening groups, garden centres, wildlife groups and trusts etc, please spread the word. [You can request laminated Asian hornet posters from the Non Native Species Secretariat (NNSS) to give to these groups].
Our Asian Hornet Action Team is getting prepared ahead of the Spring and we're printing new leaflets and ID cards for members ready for what we expect will be more sightings this year.
Here's hoping that 2019 brings us strong and healthy colonies supported by local people who, just like us, want to see honeybees and all pollinators flourish.
October 2019


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|On the Road Price
|Chambers Discount (Including Test Drive Discount)
|Chambers Price
|Customer Deposit
|38 Monthly Payments
|Amount of Credit
|Charge for Credit
|Purchase Fee
|Finance Facility Fee
|Total Amount Payable
|Optional Final Payment
|Annual Mileage
|6,000 miles
|Excess Mileage Charge
|39 Months
|APR Representative
We are pleased to announce that Chambers Ford is now part of Johnsons Cars Group!
The Ford Kuga is a smart SUV that will seamlessly integrate into your hectic life and make your time on the road that bit more comfortable and enjoyable. Carefully considered and purposeful detailing allow the Kuga to create its own stand-out appeal.
Making your life that little bit easier, the Kuga comes equipped with a hands-free tailgate to make opening and closing the boot a thing of ease. Sensing the proximity of the KeyFree key fob, you simply have to wave your foot under the rear bumper to get it open! If you are looking for a car that delivers the option for more adventure, a retractable tow bar is available to suit your needs.
Helping you to manage all driving situations, the innovative Active City Stop assists you in avoiding a collision in slow-moving traffic or when travelling at speeds up to 30mph, along with the Electronic Stability Control and anti-lock braking system, which allows you and your passengers to remain safe at all times.
Visit the Johnsons Ford dealership located in Sutton Coldfield to find out more about the adventurous Ford Kuga and take it for a test drive today. If you would prefer to find out more first, please give us a call, where a member of our helpful and friendly team will be able to answer your questions.
This is the manufacturer's list specification for this model, please verify the details when you view the vehicle. The list below will not include any additional options fitted to the vehicle or modifications.
Terms and Conditions: Finance subject to status. Subject to availability at participating dealers only on vehicles registered by 30th December 2019. Available to retail customers at participating dealers only, subject to availability on selected models only. Offers stated are cannot be used in conjunction with any other offer, discount or privilege purchase scheme. In addition, offers can not be transferred to any recent bookings, orders or purchases made before this offer. Stated offers cannot be reimbursed. There is no cash alternative to any offers stated within this page. Images and vehicles shown for illustration purposes only.
*0% APR Representative: At the end of the agreement there are 3 options: (i) Renew: Part exchange the vehicle. (ii) Retain: Pay the Optional Final Payment to own the vehicle or (iii) Return the vehicle. Further charges may be made subject to the condition or mileage of the vehicle. Terms and conditions apply. Applicants must be 18 or over. Guarantee/Indemnity may be required. Deposit allowance only available when financed with Ford Credit. All figures include vat unless otherwise stated. All figures based on 6,000 miles per annum and is for a standard no cost option paint colour, metallic and pearlescent are at extra cost.
^£1,000 Kuga Saving: customer saving (including VAT) off the Recommended on the Road price of a new Ford Kuga, that is contracted and registered between 7th and 30th December 2019. Promotion available to Retail customers only. Subject to availability. The Customer Saving is in included in this finance example.


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cadenza, colours, F, ian, in, jesus, kanyani, koper, loved, luciano, music, RARESH, Slovenia, valentio, you
Imagine a custom tailored summertime open-air get-together in the romantic setting of a historic Mediterranean city citadel wrapped in a vanguard electronic music. The celebration of laid-back open-minded people, free spirit, creative ideas, arts and culture that are setting pace to life in the territories between the Alps and Adriatic Sea. Celebrating his birthday Valentino Kanzyani is giving away two things he's best at sharing: his love and music. Headlining this unique celebration For Koper, he'll be supported by a handful of respected international artists and his personal friends Ian F, Francesco del Garda, Romanian underground superstar Raresh and the master of visuals VJ Rasta. This free entry daily event, an 8-hour back2back ride, will be held in the historic Venetian warehouse near the marina on July 27th and it starts at 5 pm.
This event is supported by City Municipiality of Koper and Radio Koper.
VALENTINO KANZYANI (si) vs RARESH (ro)
Cadenza, Jesus Loved You // Arpiar, Cocoon, Sunrise
IAN F (si) vs FRANCESCO DEL GARDA (it)
Pristaniška ulica
Koper / Capodistria
Valentino Kanzyani for Cadenza podcast – Cycle
Valentino Kanzyani for Cadenza podcast – Source
Valentino Kanzyani – Love & gratitude EP3 – Cadenza
Raresh in the mix
Ian F in the mix
Francesco del Garda in the mix


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Districting Commission
83 Broadway, Newburgh, New York 12550
Phone: (845) 569-7354
Fax: (845) 569-7370
For immediate release
February 27, 2013
Districting Commission Finalizes New
City of Newburgh Ward Boundaries
At their February 16, 2013 meeting, the Districting Commission made history by finalizing the map of the ward boundaries for the new ward system in the City of Newburgh. Chris Knasiak and Jason Nordlund co-chaired the Commission which also included Nora Cofresi, Arreda Cotten, Janet Gianopoulos, Thomas Murphy, and Rev. Steve Ruelke.
In 2011, voters approved charter amendments proposed by the 2010 Charter Review Commission providing for the creation of a ward system and a Districting Commission, and procedures for the selection and removal of the City Manager and Interim City Manager. In November 2013, voters will elect one council member from each ward.
Beginning in 2014, the City Council will consist of seven members, with two members and the Mayor elected at-large, and four members elected by ward.
A new Districting Commission will be selected every 10 years after the U.S. Census to evaluate existing wards and redistrict them as necessary so that they are in compliance with federal and state law.
"The ward system is not only a new look for the City of Newburgh," said former Mayor Nicholas Valentine, who convened the Charter Review Commissions of 2006 and 2010.
"It is a way to help insure equal representation, and that the people on the Council understand the concerns of the residents of their particular neighborhoods. I want to thank the Charter Review Commissions and the Districting Commission for their hard work, and the voters of the City of Newburgh for their overwhelming support of this historic change."
CITY PROPOSITION NO 1 and 2 Approved by the Voters Nov. 8, 2011


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Pfeiffer Men's Lacrosse Wins at Greensboro, 13-10
Greensboro, N.C. – The Pfeiffer University men's lacrosse team used a big third quarter to surge ahead of Greensboro College and hung on for 13-10 win in a USA South game.
The Basics
FINAL – Pfeiffer 13, Greensboro 10
Records -
Pfeiffer - 8-6, 5-1 USA South
Greensboro – 5-10, 0-5 USA South
Location – Greensboro, N.C. – Pride Field
John Allen led all Falcon scorers with four goals on six shots while Jacob Leone and Jared Nelson netted three goals each in the win. Nelson also added a pair of assists in the win while Leone had one. Chris Alcorn managed a goal and an assist in the win, as did Luke Cox and Spencer Stokes.
Allen opened the scoring in the first quarter with a man-up opportunity after receiving a pass from Nelson at the 14:07 mark, giving Pfeiffer the 1-0 lead. Greensboro got an unassisted goal at 6:06 to tie it at 1-1, but Spencer Stokes responded with an unassisted goal of his own at 5:41, putting Pfeiffer back on top 2-1. Greensboro scored at 5:23 to tie it at 2-2. Luke Cod added a goal just before the end of the quarter to put Pfeiffer up 3-2.
In the second quarter Greensboro held Pfeiffer scoreless and jumped ahead on a pair of goals late in the quarter.
In the third quarter, Pfeiffer notched three early goals, with Leone scoring an unassisted goal at 14:46 and Allen adding a man-up goal at 14:26. Leone scored again at 13:42 to put Pfeiffer up 6-5.
Leone scored again at 8:06 as did Allen at 2:01. Chris Alcorn scored 24 seconds before the end of the quarter to put the Falcons up 9-7 heading to the final quarter.
In the fourth, Greensboro scored at 12:05 to draw to 9-8, but three straight Falcon goals, two from Nelson and one from Allen put Pfeiffer ahead 12-8. Greensboro drew to 12-9, but Nelson scored with 2:22 left to push the lead to 13.9. Greensboro then added a goal with 32 seconds remaining to provide the 13-10 final
Up Next
The Falcons will close out their regular season on Saturday, April 20 at 7:00 p.m. with a senior night home game against William Peace.


text: gordon Posted May 10, 2007 Hello, My name is Gordon. I'm 45 and from the UK. I found this site today and have spent most of the day reading peoples stories,and experiences,it has been quite something,i myself have not yet had a full diagnosis,im still after 3 weeks waiting for skin biopsy results,i have all the signs and pains of this condition which started with a skin scaring on my right arm,then a very large bruise like wound on my leg,my local general practitioner had no idea what the problem was and I was left with no idea what was going on,i was eventualy sent to a consultant who gave me some idea and sent me for the biopsy since her diagnosis my condition has got worse both legs all up my trunk both sides slightly across the chest and down both arms,i would like to say I havent been able to ask any body about this condition as I am the second case my consultant has seen in 13 years,its the not knowing that has been frightning,thanks for a website like yours I feel a little more at ease although I have a long way to go,can somebody tell me if this condition affects the protiens in the blood,as ive been told I have a problem there sorry to go on. Many thanks, Gordon Quote Share this post Link to post Share on other sites


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Our email newsletters are a great way to receive useful information about the latest technologies and how they are affecting health organisations. Whether you are interested in our services or just interested in the latest digital developments the monthly newsletter will have something to inspire you.
Simply enter your details in the form provided below to sign up:


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Knowledge is Power!
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I am Mr Ibrahim Attah, I have something very important to shear with you. contact me through my Email Address: email@example.com. for more details.
Ibrahim Attah.
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Martin Schumm, 105, Ellis, died Tuesday, June 24, 2014, at Trego County-Lemke Long-Term Care Center, WaKeeney.
He was born Jan, 30, 1909, in Illischestie, Bukovina, Austria, to Andrew and Barbara (Bachmeier) Schum.
He married Caroline Schaus on Feb. 4, 1938, in Tulsa, Okla. She preceded him in death May 14, 2014.
He worked in the oilfields in Oklahoma and Kansas as a rough-neck in wildcat drilling from the 1930s through 1960s. He was a school custodian at Washington Grade School after the new junior high was added, and he helped Ray Eaton in his plumbing business.
He was a member of Christ Lutheran Church since 1946.
Survivors include two daughters, Beverly McLaury, Ellis, and Aleta "Lea" McCloud, Wichita; seven grandchildren, Kirk Kutina and wife, Pam, Laurie Bishop and husband, Tom, Cindi Renyer, Todd McLaury and wife, Lisa, Monica Fisher and husband, Darin, Darrin McCloud and wife, Kari, Andrea Johnson and husband, Chris; 19 great-grandchildren; 12 great-great-grandchildren; and many nieces and nephews.
He was preceded in death by his parents; his in-laws; his siblings, John, Bertha Brack and husband, Isaac, Kathryn "Katie" Coleman and husband, Earl, Ella Massier and husband, Frank, and Minnie Brown and husband, Vince; and a grandson, Clay Kutina.
Services will be at 10:30 a.m. Saturday at Keithley Funeral Chapel, 400 E. 17th, Ellis, KS 67637; burial in Mount Hope Cemetery.
Visitation will be from 5 to 7 p.m. Friday at the funeral home.
Memorials are suggested to Christ Lutheran Church in care of the funeral home.
Condolences can be sent to the family by guestbook at www.keithleyfuneralchapels.com or emailed to email@example.com.


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Two-hundred years ago the United States went to war with Great Britain to preserve its economy and its independence. With the war of 1812 underway, the United States Navy took on Great Britain's fleet and proved to be a great force of innovation while they kept the sea and America safe.
It was during the War of 1812, often referred to as the "Second War of Independence", that Francis Scott Key was inspired to write "The Star-Spangled Banner" and the Navy proved to be essential to our nation's defense by ensuring the safety of the seas. The Bicentennial Commemoration of the War of 1812 honors the legacy of the United States Navy and reminds Americans that freedom is just as important to our country today as it was two hundred years ago.
Throughout 2012 and through 2015, the U.S. Navy, U.S. Marine Corps and the U.S. Coast Guard are commemorating the War of 1812 by holding grand events throughout the country. As part of this celebration, the U.S. Navy conducts Navy Weeks across the country to share the story of the Navy with people from all over.
The United States Navy has selected Chicago as one of its Navy Week home bases for 2012. Chicago Navy Week will run in conjunction with the commemoration of the War of 1812 and week-long events will be taking place in Chicago to recognize America's Navy, August 13-20 culminating with the Chicago Air and Water Show
At Navy Pier, five active ships from the U.S. Navy, U.S. Coast Guard and Royan Canadian Navy will be docked and available for public tours, August 16 – 19. Navy Pier will also host performances by U.S. Navy bands in the Landshark Beer Garden.
For more information please visit www.navypier.com. Be sure to 'Like' Navy Pier on Facebook and follow @NavyPier on Twitter to stay connected to all the year-round fun happening at Navy Pier.


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The Pensions Authority would like to place cookies on your computer to help us make this website better. To find out more about the cookies, see our Cookie Policy.
This section provides information on the requirements for pension schemes operating 'cross-border' in Ireland.
Section 37 of the Social Welfare and Pensions Act 2005 introduced a new Part XII into the Pensions Act, 1990 to allow pension schemes to engage in cross-border activity. Directive 2003/41/EC, on the activities and supervision of institutions for occupational retirement provision (IORPs), requires that schemes registered or established in one Member State will be entitled to carry on business and accept contributions from employers based in other Member States in respect of their employees. The home Member State supervisory authority (State where the scheme is registered/authorised) is primarily responsible for supervising the scheme, however the host Member State supervisory authority (State whose social and labour law is applicable to the employment relationship between the sponsoring employer and the members) is responsible for supervising compliance with host Member State social and labour law and disclosure of information requirements.
The briefing note below on the requirements of Irish social and labour law as of 16 June 2006 has no legal status and is not to be relied on as a statement of the contents of Parts III, V, section 59B and section 59C of the Pensions Act.
Part XII requires Irish based pension schemes to apply to the Pensions Authority for authorisation before accepting any cross-border contributions. Each time a scheme wishes to accept contributions in respect of overseas employees further information must be supplied to the Authority so it can make a decision on whether the proposed arrangement is compatible with the scheme. Irish schemes engaging in cross-border activity must abide by the social and labour law and information requirements and any investment requirements applicable to any scheme members in other States.
If a pension scheme wishes to engage in cross-border activity the authorisation and approval forms below must be completed (including all relevant documentation) and submitted to:
The Pensions AuthorityVerschoyle House 28/30 Lower Mount Street Dublin 2D02 KX27
Format:DOCX|File Size:940KB
Format:DOCX|File Size:925KB


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Rory's Story Cubes Mini-Story #17
"First investigation, huh, kid?"
"Yes, captain."
"Feel like you could go in about a hundred different directions, right?"
"Yes, captain, but how—?"
"We all feel that way the first time. Think of it as a puzzle, and as you zoom in on the pieces, they all fall together. Yeah, the methodology is a little awkward, but, hey, it's not like we can go knocking on doors, right."
"No, captain."
"And this ball of rock. I can't tell how many times I've been to this backwater planet, but the brass, you know, they like messing with this species' heads. We chase their airplanes, mutilate their cattle, and the crop circles! Man, if I had a credit for every crop circle I've done, well, I could have retired a long time ago. All right, kid, since it's your first, you pick the mission."
"Well, then, I opt for a…cattle mutilation!"
"You newbies. You always go for that."


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Jigga seldom uses Twitter and when he does, it usually blows up. Today was no different. Not too long ago, the god emcee tweeted out a picture of him laying in the middle of the new Brooklyn Nets logo accompanied with an R. Kelly quote:
So what he only owns one-fifteenth of one percent? Hova was very instrumental in moving the franchise from the swamps of New Jersey into the heart of Brooklyn and is basically the face of the franchise. Just imagine how much he made from that initial one million dollar investment already. The Nets kick off their season against their cross-town rival New York Knicks at "the house that Jay built" on November 1.
The NBA preseason starts on October 5 and the regular season begins on October 30. But if you're like us, you'll be copping that NBA 2K13 next week.
[via BlackSportsOnline]


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Commodities Market Indicators: U.S. Government Data
The commodity waters can be perilous at times, and knowing how to navigate them is crucial. Keeping your eye on where the markets are heading will help you develop a winning investment strategy. Data compiled by the U.S. government is free and easy to access.
Consumer Price Index
The Consumer Price Index (CPI), compiled by the Bureau of Labor Statistics (BLS), is a statistically weighted average of a basket of goods and services purchased by consumers around the country. The CPI is the closest thing to a cost-of-living index and is sometimes used to gauge inflationary trends.
If the CPI is rising, economists start worrying that inflation is creeping up. The CPI is sometimes broken down further into the Core CPI, which excludes items like food and energy.
Comparing the CPI with the Core CPI can give you a good idea of how much consumers are spending on commodities such as energy and agricultural products.
EIA Inventory Reports
The inventory reports are released every Wednesday morning, at 10:30 a.m. EST by the Energy Information Administration (EIA), which is the statistical branch of the Department of Energy (DOE), and they detail activity in the country's energy sector.
They include a summary of weekly supply estimates, crude oil supply, and disposition rates (consumer consumption), as well as production, refinery utilization, and any movement in stock changes. The EIA petroleum inventory reports may not get wide coverage in the press, but they have a direct impact on the price of crude oil and other energy products.
Federal Funds Rate
When the financial press talks about interest rates going up or down, they're almost always referring to the Federal Funds Rate. This is the short-term interest rate at which banks charge each other overnight for Federal Reserve balances.
When the Fed wants to stimulate a sluggish economy, it tends to decrease this short term rate. On the other hand, if the Fed believes that the economy is overheating, and therefore subject to inflation, it increases this rate, which makes it more expensive to borrow money.
Gross Domestic Product
Gross Domestic Product (GDP) is essentially a measure of all the goods and services produced in a country by private consumers, the government, the business sector, and trade (exports – imports). GDP, especially per capita GDP — which measures purchasing power on an individual level — is a good indication of the likely demand for commodities.
The higher the GDP growth, the more likely a country is to spend more money on purchasing crude oil, natural gas, and other natural resources. Of course, GDP provides you with a big picture of the economic landscape and may not necessarily identify specific trends. That said, solid and growing GDP is a good measure of economic health and is a bullish indicator for commodities.
While you could theoretically analyze the GDP of all countries, looking at U.S. and Chinese GDP should be sufficient, because these two countries are the biggest purchasers of commodities such as crude oil and steel.
Non-farm Payrolls
Like the Consumer Price Index, non-farm payrolls are compiled by the Bureau of Labor Statistics. Statistically, non-farm payrolls includes the number of individuals with paid salaries employed by businesses around the country. It does not include government employees, household employees, individuals who work in the non-profit sector, and those involved in agriculture.
Non-farm payrolls include information on about 80 percent of the nation's total workforce, and this number is often used to determine unemployment levels. The non-farm payroll report is released monthly, on the first Friday of the month, and does not include total employment; rather it shows a change between the current employment levels and previous employment levels as measured by the new number of jobs that were added.
The higher the number, the stronger the economy and the more people hired by businesses — which all means that consumers have more money to spend. Although the link is indirect, higher non-farm payroll numbers can be interpreted as a bullish sign for the commodities markets.


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Salesforce has promoted Oracle's former top sales exec to be its new COO
This isn't the first COO the company has ever had. That role was previously held by George Hu, the guy that famously started at Salesforce as an intern and worked his way up to the No. 2 slot over 13 years.
Hu left Salesforce the end of 2014 to found his own startup, Peer, (with the blessing, and financial backing, of Salesforce CEO Marc Benioff, who is a huge angel investor).Meanwhile, Block has done extremely well at Salesforce since he left his old alma mater, Oracle, about a year before joining Salesforce. Block had been head of North American sales.
He left Oracle after a controversial bunch of instant messages and e-mails from him surfaced during a lawsuit between Oracle and HP, some of them saying less-than-nice things about Mark Hurd's strategy. Hurd was a president for Oracle at the time in charge of sales (and has since been promoted to a joint CEO role, a job he shares with Safra Catz).
In 2014, Benioff called Hurd's decision to dismiss Block "the biggest mistake Mark Hurd ever made." (Sources close to Oracle told Business Insider at the time, that decision to let Block go wasn't entirely Hurd's alone.)
Salesforce and Oracle are fierce rivals since Oracle has turned its attention towards cloud computing, where Oracle is a relative latecomer. Oracle's executive chairman Larry Ellison frequently names Salesforce as one of his biggest competitors.
Meanwhile, Block found his calling running sales at Salesforce. In the time he's been there, Salesforce's revenues and bookings have skyrocketed.
The company has promised revenues will hit over $6.6 billion for its current fiscal year when it announces those results probably later this month. And it's on track to hit $8 billion next year, it says, up from $3.05 billion in 2013 the year Block joined. Benioff says the company is quickly marching toward $10 billion, too, and names Block a key player in its success.


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Promising Candidates, Positive Results: Best Sales and Marketing Recruitment Agency in the UK
Dec 30, 2017
Simon Sinek was absolutely correct when he said, "If you hire people just because they can do a job, they'll work for your money. But, if you hire people who believe what you believe, they'll work for you with blood and sweat and tears." Keeping the same in mind while looking for the best sales and marketing professionals will help you make the best hire. However, if you lack time as well as the expertise, relying on a recruitment agency for finding the perfect sales and marketing talent would be fruitful. Recruitment experts, being aware of the difference between the job profiles in the fields of sales and marketing, would strive their best in finding the suitable candidates. What's best is that owing to their detailed knowledge of the two sectors (sales and marketing), recruitment experts won't repeat the mistakes that most of the companies do while hiring sales and marketing candidates.
When it comes to finding the best sales and marketing recruitment agency in the UK, organisations rely on Jefferson Maguire. Backed by a team of skilled and proficient headhunting professionals, we are experts at tracking down and recruiting the best candidates for sales and marketing teams for countless companies. With us at bay, there's no need for you to look for sales and marketing recruitment agencies in London. Committed to providing our clients with ideal candidates as per their job vacancy requirements, we, a firm of International Headhunters, are preferred by all in distinct sectors.
So, put an end to your search for sales and marketing professionals for your team. Give us a chance to prove our caliber and provide you with top quality sales and marketing candidates!
Executive Headhunters Specialising in Many Sectors
Know who you need but don't know how to find them?
Talk to one our our experienced headhunters to track down your perfect personLets Talk
My personal guarantee to you...
I will personally guarantee that whether a client or candidate you will receive a truly exceptional experience using our service.
David Pike
MD, Jefferson Maguire


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After excitedly seeing the launch of MAC' limited edition 'To The Beach' range; a beautiful seaside inspired collection, with sultry summer golden shades that would be a welcome addition to any holiday make up bag, I placed my order with a kind friend who was doing a small MAC haul.
Then the gloomy call came from my disgruntled friend, not only are most of these items in this edition nowhere to be found in any London stores, with the High-Lighter in 'Marine Life' that I had my heart set on totally sold out both in store and on-line. [quiet sob]
This is my plea to MAC……. please replenish stock most urgently or I will have to take to the black market in sheer desperation.