Low Mr Research Articles

Sustainable Construction Case History: Fly Ash Stabilization of Recycled Asphalt Pavement Material

A case history is described where Class C fly ash was used to stabilize recycled pavement material (RPM) during construction of a flexible pavement in Waseca, MN, USA. The project consisted of pulverizing the existing hot-mix asphalt (HMA), base, and subgrade to a depth of 300 mm to form RPM, blending the RPM with fly ash (10% by dry weight) and water, compacting the RPM, and placement of a new HMA surface. California bearing ratio (CBR), resilient modulus (Mr), and unconfined compression (qu) tests were conducted on the RPM alone and the fly ash stabilized RPM (SRPM) prepared in the field and laboratory to evaluate how addition of fly ash improved the strength and stiffness. After 7 days of curing, SRPM prepared in the laboratory had CBR ranging between 70 and 94, Mr between 78 and 119 MPa, and qu between 284 and 454 kPa, whereas the RPM alone had CBR between 3 and 17 and Mr between 46 and 50 MPa. Lower CBR, Mr, and qu were obtained for SRPM mixed in the field relative to the SRPM mixed in the laboratory (64% lower for CBR, 25% lower for Mr, and 50% lower for qu). In situ falling weight deflectometer testing conducted 1 year after construction showed no degradation in the modulus of the SRPM, even though the SRPM underwent a freeze–thaw cycle. Analysis of leachate collected in the lysimeter showed that concentrations of all trace elements were below USEPA maximum contaminant levels.

Immunomodulatory activity of acidic polysaccharides isolated from Tanacetum vulgare L.

Tanacetum vulgare L. (Tansy) has been extensively used in folk medicine for treatment of a variety of medical disorders. In the present study, we isolated and purified four acidic polysaccharide fractions (designated T-I to T-IV) from Tansy florets by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, anion-exchange, and size-exclusion chromatography. The average Mr of fractions T-I through T-IV was estimated to be 326, 151, 64 and 9 kDa, respectively, as determined by high performance size-exclusion chromatography analysis. Sugar composition analysis revealed that Tansy polysaccharides consisted primarily of galacturonic acid, galactose, arabinose, and rhamnose. Fractions T-II through T-IV contained an arabinogalactan type II structure, as determined by reaction with Yariv reagent. High Mr fractions T-I and T-II exhibited potent macrophage/monocyte-activating activity, enhancing production of reactive oxygen species (ROS), nitric oxide (NO), and tumor necrosis factor α (TNF-α) by J774.A1 murine macrophages, and activating nuclear factor κB (NF-κB) in THP-1 human monocytes. In addition, Tansy polysaccharides stimulated human neutrophil function by greatly enhancing neutrophil myeloperoxidase (MPO) release. Furthermore, the low Mr fraction T-IV had potent complement-fixing activity, which may also contribute to the anti-inflammatory and would-healing properties of Tansy extracts. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Tansy extracts, and support the concept of using Tansy polysaccharides as an immunotherapeutic adjuvant.

Effects of internal browning and watercore on low field (5.4 MHz) proton magnetic resonance measurements of T2 values of whole apples

Apples with internal browning and/or watercore along with unaffected apples were tested using a low frequency (5.4 MHz) proton magnetic resonance ( 1H MR) sensor. A three-term exponential model was fit to the spin–spin relaxation ( T 2) curves obtained from individual apples. The contributions from terms of the model associated with extracellular spaces, a (ca. 16 ms) and the cytoplasm, b (ca. 300 ms) increased with the severity of internal browning ( r 2 = 0.7, 0.9, respectively). Conversely, the contribution of the term associated with water in the vacuoles, c (ca. 1260 ms) decreased as the severity of internal browning increased ( r 2 = 0.9). The changes in the model parameters were different for apples having only internal browning compared to apples with both moderate watercore and slight or no internal browning and the differences were statistically significant ( P < 0.05). Percent total soluble solids (TSS) in the apples was not affected by the severity of the internal browning for apples with moderate watercore while it increased with severity of internal browning for apples without moderate watercore. In addition, the density of the apples decreased as the contribution of the term associated with water in the vacuoles increased. Finally, two of the model parameters were significantly correlated with TSS in unaffected apples and one parameter was significantly correlated with TSS in apples with moderate watercore and no internal browning ( r 2 ≅ 0.7). The results of this study suggest that when a three-term exponential model is fit to the T 2 relaxation curve from a low field 1H MR sensor, the changes in the parameters may give an indication of the presence and severity of internal browning as well as the presence of watercore in the apple.

N-Glycosylation of the Human κ Opioid Receptor Enhances Its Stability but Slows Its Trafficking along the Biosynthesis Pathway

We examined glycosylation of FLAG-hKOR expressed in CHO cells and determined its functional significance. FLAG-hKOR was resolved as a broad and diffuse 55-kDa band and a less diffuse 45-kDa band by immunoblotting, indicating that the receptor is glycosylated. Endoglycosidase H cleaved the 45-kDa band to approximately 38 kDa but did not change the 55-kDa band, demonstrating that the 45-kDa band is N-glycosylated with high-mannose or hybrid-type glycan. Peptide-N-glycosidase F digestion of solubilized hKOR or incubation of cells with tunicamycin resulted in two species of 43 and 38 kDa, suggesting that the 43-kDa band is O-glycosylated. FLAG-hKOR was reduced to lower Mr bands by neuraminidase and O-glycosidase, indicating that the hKOR contains O-linked glycan. Mutation of Asn25 or Asn39 to Gln in the N-terminal domain reduced the Mr by approximately 5 kDa, indicating that both residues were glycosylated. The double mutant hKOR-N25/39Q was resolved as a 43-kDa (mature form) and a 38-kDa (intermediate form) band. When transiently expressed, hKOR-N25/39Q had a lower expression level than the wild type. In CHO cells stably expressing the hKOR-N25/39Q, pulse-chase experiments revealed that the turnover rate constants (ke) of the intermediate and mature forms were approximately 3 times those of the wild type. In addition, the maturation rate constant (ka) of the 43-kDa form of hKOR-N25/39Q was 6 times that of the mature form of the wild type. The hKOR-N25/39Q mutant showed increased agonist-induced receptor phosphorylation, desensitization, internalization, and downregulation, without changing ligand binding affinity or receptor-G protein coupling. Thus, N-glycosylation of the hKOR plays important roles in stability and trafficking along the biosynthesis pathway of the receptor protein as well as agonist-induced receptor regulation.

Effect of Interfacial Spin Flip and Momentum Scattering on Magnetoresistance

In this paper, we have developed a model and performed a numerical analysis using the spin-drift diffusion method to study the effect of interfacial spin scattering on the magnetoresistance of a simple trilayer spin-valve structure. We have modelled the interfaces as ultra-thin layers, in the limit of the layer thickness approaching zero. Our analysis is focused on three important interfacial parameters, i.e., interfacial resistance (RI), interfacial spin selectivity (gamma), and interfacial spin-flip parameter (zeta). We compared the relative contribution of bulk scattering (BS) and interfacial scattering (IS) to the overall MR. We have noticed that when gamma is greater (smaller) than a critical value, gammaC, then MR increases with increasing (decreasing) RI. Contrary to general expectation, larger ferromagnetic (FM) resistivity results in lower MR if gamma>gammaC. Finally, we have shown the effect of zeta on MR. At high (low) zeta, which signifies spin coherence at the interface is conserved (depolarized), MR increases (decreases). We have also noted the competition between gamma and zeta in contributing towards overall MR. We have also shown that the negative effect on MR due to spin flip can be minimized by using higher RI

Interorganelle Trafficking of Ceramide Is Regulated by Phosphorylation-dependent Cooperativity between the PH and START Domains of CERT

The synthesis and transport of lipids are essential events for membrane biogenesis. However, little is known about how intracellular trafficking of lipids is regulated. Ceramide is synthesized at the endoplasmic reticulum (ER) and transported by the ceramide transfer protein CERT to the Golgi apparatus, where it is converted to sphingomyelin. CERT has a phosphoinositide-binding pleckstrin homology (PH) domain for Golgi-targeting and a lipid transfer START domain for intermembrane transfer of ceramide. We here show that CERT receives multiple phosphorylations at a serine-repeat motif, a possibe site for casein kinase I, and that the phosphorylation down-regulates the ER-to-Golgi transport of ceramide. In vitro assays show that the phosphorylation induces an autoinhibitory interaction between the PH and START domains and consequently inactivates both the phosphoinositide binding and ceramide transfer activities of CERT. Loss of sphingomyelin and cholesterol from cells causes dephosphorylation of CERT to activate it. The cooperative control of functionally distinct domains of CERT is a novel molecular event to regulate the intracellular trafficking of ceramide.

Micromagnetism and magnetotransport properties of micron-sized epitaxialCrO2(100)wires

We report a detailed study of the magnetic domain configurations and the magnetization reversal in epitaxial CrO2100 wires 0.5– 5 m widths patterned along different crystallographic directions. Magnetic force microscopy imaging in zero field reveals single domain states for wires fabricated along the magnetic easy axis, while wires perpendicular to the magnetic easy axis exhibit a stripe domain configuration. The behavior in an applied field and the switching of the magnetization is probed by magnetoresistance MR measurements. Depending on the orientation of the wire with respect to the easy and hard magnetocrystalline anisotropy axes and the field direction, distinctly different reversal modes are observed including inhomogeneous magnetization configurations. These modes and configurations can be explained taking into account the interplay between the different anisotropy terms. MR measurements at variable temperatures demonstrate that the low temperature MR response is dominated by intergrain-tunneling magnetoresistance, while at elevated temperatures it is superseded by the anisotropic MR.

Hydrodynamic sorting and transport of terrestrially derived organic carbon in sediments of the Mississippi and Atchafalaya Rivers

Over the course of two years, four cruises were conducted at varying levels of discharge in the lower Mississippi and Atchafalaya Rivers (MR and AR) where grab samples were collected from sand- and mud-dominated sediments. The tetramethylammonium hydroxide (TMAH) thermochemolysis method was used to determine sources of terrestrially derived organic carbon (OC) in these two sediment types, to examine the effects of hydrodynamic sorting on lignin sources in river sediments. Average lignin concentrations in the lower MR were 1.4 ± 1.1 mg gOC −1 at English Turn (ET) and 10.4 ± 27.4 mg gOC −1 at Venice. Using these concentrations, annual lignin fluxes to the Gulf of Mexico, from tidal and estuarine mud remobilization at ET and Venice, were 3.1 ± 2.5 × 10 5 kg and 11.4 ± 30.0 × 10 5 kg, respectively. Much of the lignin-derived materials in muddy sediments appeared to be derived from non-woody grass-like sources – which should decay more quickly than the woody materials typically found in the sandy deposits. The average total OC% (1.93 ± 0.47) of English Turn sands yields an annual flux of 0.34 ± 0.09 × 10 9 kg. Lignin flux in the English Turn sands (3.6 ± 2.6 mg gC −1) using the numbers above would be 12.2 ± 9.4 × 10 5 kg. The extensive amounts of sand-sized woody materials (coffee-grinds) found in the sandy sediments in both the AR and MR are likely derived from woody plant materials. This is the first time it has been demonstrated that sandy sediments in the MR provide an equally important pathway (compared to muds) for the transport of terrestrially derived organic matter to the northern Gulf of Mexico. Using the AR average %OC in sand (1.16 ± 0.72), we estimated an annual flux of OC to the shelf of 0.13 ± 0.07 × 10 9 kg. Lignin flux for AR sands was estimated to be 12.4 ± 12.1 × 10 5 kg. Despite the high error associated with these numbers, we observe for the first time that the flux of lignin in sandy sediments in the AR to the northern Gulf of Mexico is comparable to that found in the MR. These results further support the likelihood of grain-size related hydrodynamic sorting of terrestrially derived organic carbon in the lower Mississippi and Atchafalaya Rivers, suggesting that there is a distinct sandy sediment organic fraction contributed by major rivers to the global carbon cycle.

An NMR study on internal browning in pears

Internal browning in pears ( Pyrus communis L. cv. Blanquilla) has been studied by NMR and MRI in order to develop a non-destructive procedure for on-line disorder identification. For NMR relaxometry, disordered tissue shows higher transverse relaxation rates compared to sound tissue, especially at higher magnetic field strength and for long pulse spacing. Membrane alteration and therefore tissue disintegration, as well as water evaporation, appear to be the main causes of this response. Correlation between relaxation times and diffusion showed that the proton pools in disordered tissue are grouped into a smaller number of populations compared to sound tissue, also highlighting cell decompartmentation in disordered tissue. At a macroscopic level, fast low angle shot MR images, effective transverse relaxation-weighted (TR 11 ms and TE 3.7 ms) and proton density-weighted (TR 7.6 ms and TE 2.5 ms), were acquired for pears at a rate of 54 mm/s. Images have been discriminated for internal breakdown according to histogram characteristics. Up to 94 and 96% of pears, respectively, were correctly classified in the former and the latter type of images. In this study a minimum value of 12% of tissue affected by breakdown was always clearly identified.

Preoperative MR Angiography in Free Fibula Flap Transfer for Head and Neck Cancer: Clinical Application and Influence on Surgical Decision Making

We review the fibular free flap surgical procedure to illustrate the usefulness of preoperative lower limb MR angiography and to show how calf vascular anatomy on MR angiography affects patient surgical management. With its high positive predictive value and sensitivity, preoperative MR angiography can improve the chances of a successful outcome at the recipient mandibular site. It provides the reconstructive surgeon with a road map, revealing vascular anomalies or disease that could alter or contraindicate surgery.

The Hemolymph of the Ascidian Styela plicata (Chordata-Tunicata) Contains Heparin inside Basophil-like Cells and a Unique Sulfated Galactoglucan in the Plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

First results of floating cage culture of the African catfish Heterobranchus longifilis Valenciennes, 1840: Effect of stocking density on survival and growth rates

The main constraint of the culture of the African catfish Heterobranchus longifilis remains the high mortality rate due mainly to cannibalism. Heterobranchus longifilis is generally cultured in earth ponds, tanks and enclosures. Floating cage aquaculture can be an alternative system to these traditional techniques as it may allow predation control. Therefore, the purpose of this study was to test the effects of floating cages (1 m 3) on survival and growth parameters of H. longifilis at different stocking densities in man-made Lake Ayame. In a first experiment which started with fish of 0.8 ± 0.1 g initial mean weight, five stocking densities (50, 100, 200, 500 and 1000 fish/m 3) were tested during a 90-day experiment. The results showed that unlike final mean weight (Wf) and mean daily weight gain (Mdwg), weight variation coefficient (final Cv), cannibalism (Cr), mortality (Mr) and survival (Sr) were density dependent: best results of survival, cannibalism and mortality (68.0 ± 1.5%, 18.0 ± 2.2% and 14.0 ± 1.5% respectively) were recorded at the lowest density (50 fish/m 3). In a second experiment that began with fish of 13.4 ± 0.5 g (Wi) and lasted for 270 days, the stocking densities tested were 6, 12, 25, 50 and 100 fish/m 3. Results of this experiment showed that all the parameters [Mr, Cr, Sr, final Cv and apparent feed conversion ratio (Afcr)] were influenced by the stocking densities. Higher Wf, Mdwg and Sr and lower final Cv, Cr, Mr, and Afcr were recorded at the lowest stocking densities (6 and 12 fish/m 3). Cage culture of H. longifilis results in a higher Sr, but a lower Mdwg compared to those usually recorded in traditional rearing systems. However, this low production potential of H. longifilis could be increased by the improvement of rearing conditions (feeding systems, culture management) as noted in other species.

Chemistry and genotoxicity of caramelized sucrose

Caramelization of a 1% sucrose solution at 180 degrees C accompanied characteristic changes in pH, Mr, UV-absorbance, and fluorescence values as well as increased reducing power activity after 40-60 min. Similar changes occurred to sucrose heated at 150 degrees C, after 150-240 min. Bioactivity of caramelized sucrose samples was tested for mutagenic activity, using Salmonella typhimurium strains TA-98 and TA-100, respectively, as well as the Saccharomyces D7 yeast strain for mitotic recombination and Chinese hamster ovary cells (CHO) to assess clastogenicity. Caramelized sucrose expressed no mutagenicity in the TA-98 strain, but gave positive (p < 0.05) results with the TA-100, base-pair substitution strain. Similarly, mitotic recombination in the Saccharomyces D7 yeast strain and clastogenic activity in CHO cells were induced when exposed to caramelized sucrose. In the all cases, preincubation with S-9 reduced (p < 0.05) the mutagenic activities of caramelized sucrose. Fractionation of the caramelized sucrose into volatile and nonvolatile compounds was performed and tested for clastogenicity using CHO cells. Volatile components contributed approximately 10% to total clastogenicity, which was enhanced by the presence of S-9. Nonvolatile components recovered, consisting of relatively lower Mr, gave highest (p < 0.05) clastogenic activity, denoting that higher Mr caramel colors are relatively free of this property.

The Value of Low Field intensity MR Grandient Echo and Inhibition Imange Formantion in the Diagnosis of the Injury of Knee-For 120Cases

目的 探讨低场强磁共振梯度回波和脂肪抑制成像在膝关节损伤中的临床应用.方法 120例患者经低场强MR不同序列检查,显示半月板、韧带、关节囊、关节软骨及关节面骨质等受损情况.结果 低场强MRI常规序列加脂肪抑制和梯度回波序列对内外侧半月板损伤,前、后交叉韧带,侧副韧带撕裂及关节软骨和骨质损伤的显示率高,诊断准确可靠,是膝关节损伤最佳的检查手段。

Spatially encoded pulse sequences for the acquisition of high resolution NMR spectra in inhomogeneous fields

We have recently proposed a protocol for retrieving nuclear magnetic resonance (NMR) spectra based on a spatially-dependent encoding of the MR interactions. It has also been shown that the spatial selectivity with which spins are manipulated during such encoding opens up new avenues towards the removal of magnetic field inhomogeneities; not by demanding extreme B o field uniformities, but rather by compensating for the dephasing effects introduced by the field distribution at a radiofrequency excitation and/or refocusing level. The present study discusses in further detail a number of strategies deriving from this principle, geared at acquiring both uni- as well as multi-dimensional spectroscopic data at high resolution conditions. Different variants are presented, tailored according to the relative sensitivity and chemical nature of the spin system being explored. In particular a simple multi-scan experiment is discussed capable of affording substantial improvements in the spectral resolution, at nearly no sensitivity or scaling penalties. This new compensation scheme is therefore well-suited for the collection of high-resolution data in low-field systems possessing limited signal-to-noise ratios, where magnetic field heterogeneities might present a serious obstacle. Potential areas of applications of these techniques include high-field in vivo NMR studies in regions near tissue/air interfaces, clinical low field MR spectroscopy on relatively large off-center volumes difficult to shim, and ex situ NMR. The principles of the different compensation methods are reviewed and experimentally demonstrated for one-dimensional inhomogeneities; further improvements and extensions are briefly discussed.

Quantification of p16 methylation in serum DNA as a new diagnostic tool for colorectal cancer

10050 Background: The incidence of colorectal cancer (CRC), one of the commonest malignancies worldwide, is still increasing. Despite advances in the diagnostic procedure, a large number of patients with CRC still presents with advanced disease. More sensitive and efficient diagnostic technique would make an impact on the prognosis of CRC patients through improving their compliance to the screening program, hence allowing earlier detection of the disease. To test the hypothesis that p16 methylation may serve as a marker for the diagnosis of CRC, we quantified the methylation levels of p16 in the serum DNA of CRC patients. Methods: Fresh specimens of 168 CRC and corresponding noncancerous tissues were obtained surgically at the Department of Surgery II, Nagoya University Graduate School of Medicine, together with the corresponding serum samples obtained 1 week prior to surgery. We defined the p16 methylation rate (p16 MR, in %) as follows: CM/(CM + CU) × 100%. CM is the concentration of methylated p16 sequences and CU is the concentration of unmethylated p16 sequences measured by quantitative methylation-specific PCR (Q-MSP) after bisulfite conversion. The relationship between the p16 MR and clinicopathologic findings were evaluated. Results: Aberrant p16 promoter methylation was found in 59% (99 of 168) of surgically resected CRC tissues. None of the corresponding normal tissues had methylated p16 sequences. 37% (37 of 99) serum samples of the CRC patients with tumoral p16 methylation had the same alterations. p16 MRs of 99 serum samples with tumoral p16 methylation were 0 to 68.9 (mean was 5.43±11.01). p16 MRs were significantly correlated with lymph node metastasis (P=0.001), lymphatic invasion (P=0.001) and venous invasion (P=0.020) of CRCs. p16 MRs increased significantly with tumor stage [stage I : 0.94 ± 1.68, stage II : 2.33 ± 5.19, stage III : 8.49 ± 14.22, stage IV : 10.03 ± 14.27 (P = 0.021)]. Moreover, the survival of patients with low p16 MR was significantly superior to those with high p16 MR (P=0.006). Conclusions: These results suggest that quantification of p16 methylation in the serum DNA might be a novel diagnostic tool and a prognostic factor for CRC. No significant financial relationships to disclose.

Seasonal changes in the abundance and composition of plant pigments in particulate organic carbon in the lower Mississippi and Pearl Rivers

Plant pigments in particulate organic carbon were examined in the lower Mississippi and Pearl Rivers (U.S.), along with physical variables and nutrients to study seasonal changes in the abundance and composition of phytoplankton. Water samples were collected monthly from September 2001 to August 2003 in the lower Mississippi River (MR; no samples were taken in February 2002) and from August 2001 to July 2003 in the Pearl River (PR). High concentrations of total suspended solids (TSS), nutrients, and chlorophylla (chla; dominated by diatoms) were observed in the lower MR. The smaller blackwater PR was characterized by lower nutrients and chla, higher ultraviolet absorbance, and a phytoplankton biomass dominated by chlorophytes. Chla concentrations in the lower MR was high in summer low-flow periods and also during interims of winter and spring, and did not couple with physical variables and nutrients, likely due to a combination of in situ production and inputs from reservoirs, navigation locks and oxbow lakes in the upper MR and Missouri River. Chla concentrations in the PR was only high in summer low-flow periods and were controlled by temperature and concentrations of chromophoric dissolved organic matter 9CDOM). The high, diatom-dominated phytoplankton biomass in the lower MR was likely the result of decreasing TSS (increased damming in the watershed) and increasing nutrients (enhanced agricultural runoff) over the past few decades. Lower phytoplankton biomass (dominated by chlorophytes) in the PR was likely linked with intense shading by CDOM and lower availability of nutrient inputs. An increase in the relative importance of phytoplankton biomass in large turbid rivers, such as the MR, could have significant effects on the age and lability of riverine organic matter entering the ocean, the stoichiometric balance of nutrients delivered to coastal margins, and the sequestration of atmospheric CO2 in these dynamic regions.

Enolase Activates Homotypic Vacuole Fusion and Protein Transport to the Vacuole in Yeast

Membrane fusion and protein trafficking to the vacuole are complex processes involving many proteins and lipids. Cytosol from Saccharomyces cerevisiae contains a high Mr activity, which stimulates the in vitro homotypic fusion of isolated yeast vacuoles. Here we purify this activity and identify it as enolase (Eno1p and Eno2p). Enolase is a cytosolic glycolytic enzyme, but a small portion of enolase is bound to vacuoles. Recombinant Eno1p or Eno2p stimulates in vitro vacuole fusion, as does a catalytically inactive mutant enolase, suggesting a role for enolase in fusion that is separate from its glycolytic function. Either deletion of the non-essential ENO1 gene or diminished expression of the essential ENO2 gene causes vacuole fragmentation in vivo, reflecting reduced fusion. Combining an ENO1 deletion with ENO2-deficient expression causes a more severe fragmentation phenotype. Vacuoles from enolase 1 and 2-deficient cells are unable to fuse in vitro. Immunoblots of vacuoles from wild type and mutant strains reveal that enolase deficiency also prevents normal protein sorting to the vacuole, exacerbating the fusion defect. Band 3 has been shown to bind glycolytic enzymes to membranes of mammalian erythrocytes. Bor1p, the yeast band 3 homolog, localizes to the vacuole. Its loss results in the mislocalization of enolase and other vacuole fusion proteins. These studies show that enolase stimulates vacuole fusion and that enolase and Bor1p regulate selective protein trafficking to the vacuole.

Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis

This study was conducted to evaluate monthly changes in the ram seminal plasma protein profile using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with a polyacrylamide linear gradient gel. Likewise, comparative analyses of the protein composition of ovine seminal plasma (SP) from ejaculates obtained along the year, and its relationship with sperm motility, viability and concentration of ejaculate were carried out. Western-blot analysis was performed to specifically detect P14, a ram SP protein postulated to be involved in sperm capacitation and gamete interaction [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539–49], and its variations along the year have also been established. The experiment was carried out from May 2003 to April 2004, with nine Rasa Aragonesa rams. Ejaculates obtained every 2 days were pooled and used for each assay, to avoid individual differences, and three two-dimensional SDS-PAGE gels were run for each month. The high resolution of the gradient gel allowed the image analysis software to detect around 252 protein spots, with pIs ranging from 4.2 to 7.6, and molecular weight (Mr) from 12.5 to 83.9kDa. Four protein spots (1, 2, 3 and 4) of low Mr (15.1, 15.7, 15.9 and 21.0kDa) and acidic pI (5.9, 5.3, 5.7 and 6.6), respectively, had the highest relative intensity in the SP map (11.2, 9.3, 4.7 and 7.7%, respectively). Spot 3 was more abundant (P<0.05) from May to December, and negatively correlated (P<0.05, r=−0.34) with sperm viability and concentration (P<0.05, r=0.36). Another 12 protein spots also had significant quantitative differences (P<0.05) along the year, and 17 protein spots, which correlated with some seminal quality parameter, did not show quantitative monthly changes. Western-blot analysis indicated that spots 1 and 2 reacted with the anti-P14 antibody, raised against the P14 band (approximate Mr 14kDa) of ram SP. This indicates that spots 1 and 2 are similar to RSP15 [Bergeron A, Villemure M, Lazure C, Manjunath P. Isolation and characterization of the major proteins of ram seminal plasma. Mol Reprod Dev 2005;71:461–70], bovine PDC-109 [Esch FS, Ling NC, Bohlen P, Ying S, Guillemin R. Primary structure of PDC-109, a major protein constituent of bovine seminal plasma. Biochem Biophys Res Commun 1983;113:861–7] (also called BSP A1/A2 [Manjunath P, Sairam MR. Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma. Biochem J 1987;241:685–92]) and goat GSP-14/15kDa [Villemure M, Lazure C, Manjunath P. Isolation and characterization of gelatine-binding proteins from goat seminal plasma. Reprod Biol Endocrinol 2003;1:39], based on our previous results on the P14 amino acid sequence [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539–49].

A Critical Coiled Coil Motif in the Small Terminase, gp16, from Bacteriophage T4: Insights into DNA Packaging Initiation and Assembly of Packaging Motor

Double-stranded DNA packaging in bacteriophages is driven by one of the most powerful force-generating molecular motors reported to date. The phage T4 motor is composed of the small terminase protein, gpl6 (18kDa), the large terminase protein, gp17 (70kDa), and the dodecameric portal protein gp20 (61kDa). gp16, which exists as an oligomer in solution, is involved in the recognition of the viral DNA substrate, the very first step in the DNA packaging pathway, and stimulates the ATPase and packaging activities associated with gp17. Sequence analyses using COILS2 revealed the presence of coiled coil motifs (CCMs) in gp16. Sixteen T4-family and numerous phage small terminases show CCMs in the corresponding region of the protein, suggesting a common structural and functional theme. Biochemical properties such as reversible thermal denaturation and analytical gel filtration data suggest that the central CCM-1 is critical for oligomerization of gp16. Mutations in CCM-1 that change the hydrophobicity of key residues, or pH 6.0, destabilized coiled coil interactions, resulting in a loss of gp16 oligomerization. The gp16 oligomers are in a dynamic equilibrium with lower M(r) intermediate species and monomer. Monomeric gp16 is unable to stimulate gp17-ATPase, an activity essential for DNA packaging, while conversion back into oligomeric form restored the activity. These data for the first time defined a CCM that is critical for structure and function of the small terminase. We postulate a packaging model in which the gp16 CCM is implicated in the regulation of packaging initiation and assembly of a supramolecular DNA packaging machine on the viral concatemer.