Purification of N-acetyllactosamine-binding activity from the porcine sperm membrane: possible involvement of an ADAM complex in the carbohydrate-binding activity of sperm

Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads-one recognizes N-acetyllactosamine (Galβ1-4GlcNAc-), and the other recognizes the Lewis X structure (Galβ1-4(Fucα1-3)GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane.     

Does canopy mean nitrogen concentration explain variation in canopy light use efficiency across 14 contrasting forest sites?

The maximum light use efficiency (LUE?=?gross primary production (GPP)/absorbed photosynthetic photon flux density (aPPFD)) of plant canopies has been reported to vary spatially and some of this variation has previously been attributed to plant species differences. The canopy nitrogen concentration [N] can potentially explain some of this spatial variation. However, the current paradigm of the N-effect on photosynthesis is largely based on the relationship between photosynthetic capacity (A(max)) and [N], i.e., the effects of [N] on photosynthesis rates appear under high PPFD. A maximum LUE-[N] relationship, if it existed, would influence photosynthesis in the whole range of PPFD. We estimated maximum LUE for 14 eddy-covariance forest sites, examined its [N] dependency and investigated how the [N]-maximum LUE dependency could be incorporated into a GPP model. In the model, maximum LUE corresponds to LUE under optimal environmental conditions before light saturation takes place (the slope of GPP vs. PPFD under low PPFD). Maximum LUE was higher in deciduous/mixed than in coniferous sites, and correlated significantly with canopy mean [N]. Correlations between maximum LUE and canopy [N] existed regardless of daily PPFD, although we expected the correlation to disappear under low PPFD when LUE was also highest. Despite these correlations, including [N] in the model of GPP only marginally decreased the root mean squared error. Our results suggest that maximum LUE correlates linearly with canopy [N], but that a larger body of data is required before we can include this relationship into a GPP model. Gross primary production will therefore positively correlate with [N] already at low PPFD, and not only at high PPFD as is suggested by the prevailing paradigm of leaf-level A(max)-[N] relationships. This finding has consequences for modelling GPP driven by temporal changes or spatial variation in canopy [N].     

Detection of Torque teno sus virus types 1 and 2 by nested polymerase chain reaction in sera of sows at parturition and of their newborn piglets immediately after birth without suckling colostrum and at 24 hr after suckling colostrum

This study was performed to clarify the sow-to-fetus transmission pathway of Torque teno sus virus (TTSuV) types 1 (TTSuV1) and 2 (TTSuV2). For this purpose, detection of TTSuV1 and TTSuV2 (TTSuVs) in sera of 6 sows (Sows 1-6) at parturition and in sera of their newborn piglets immediately after birth without suckling colostrum was performed by nested polymerase chain reaction (nPCR). These sows were bred using semen that had tested negative for TTSuVs. In a TTSuV1- and TTSuV2-positive sow (Sow 1), TTSuV1 and TTSuV2 were detected in 4 and 5 of 12 newborn littermates, respectively. In a TTSuV1-positive sow (Sow 2), TTSuV1 was detected in 1 of 8 newborn littermates. In 4 TTSuV1- and TTSuV2-negative sows (Sows 3-6), TTSuV1 was detected in 6 out of the 25 newborn piglets of 3 sows (Sows 3-5), while TTSuVs were not detected in all 13 piglets of 1 sow (Sow 6). In addition, to investigate the possibility of a sow-to-piglet transmission pathway of TTSuV via colostrum, TTSuV1 and TTSuV2 in sera of 12 newborn piglets from Sows 1-3 were examined by nPCR. Immediately after birth without suckling colostrum, TTSuV1 and TTSuV2 were not detected in 10 and 8 of 12 newborn piglets, respectively; however, at 24 hr after suckling colostrum, TTSuV1 was detected in 6 piglets, while TTSuV2 was not detected in any piglets. These results confirmed the existence of a sow-to-fetus transmission pathway of TTSuV during normal pregnancy and suggested a possibility of sow-to-piglet transmission of TTSuV via colostrum.     

Glycosylated Porphyra-334 and Palythine-Threonine from the Terrestrial Cyanobacterium Nostoc commune

Mycosporine-like amino acids (MAAs) are water-soluble UV-absorbing pigments, and structurally different MAAs have been identified in eukaryotic algae and cyanobacteria. In this study novel glycosylated MAAs were found in the terrestrial cyanobacterium Nostoc commune (N. commune). An MAA with an absorption maximum at 334 nm was identified as a hexose-bound porphyra-334 derivative with a molecular mass of 508 Da. Another MAA with an absorption maximum at 322 nm was identified as a two hexose-bound palythine-threonine derivative with a molecular mass of 612 Da. These purified MAAs have radical scavenging activities in vitro, which suggests multifunctional roles as sunscreens and antioxidants. The 612-Da MAA accounted for approximately 60% of the total MAAs and contributed approximately 20% of the total radical scavenging activities in a water extract, indicating that it is the major water-soluble UV-protectant and radical scavenger component. The hexose-bound porphyra-334 derivative and the glycosylated palythine-threonine derivatives were found in a specific genotype of N. commune, suggesting that glycosylated MAA patterns could be a chemotaxonomic marker for the characterization of the morphologically indistinguishable N. commune. The glycosylation of porphyra-334 and palythine-threonine in N. commune suggests a unique adaptation for terrestrial environments that are drastically fluctuating in comparison to stable aquatic environments.     

Turbid water induces refuge behaviour of a commercially important ayu: A field experiment for interstream movement using multiple artificial streams

Riverine systems are inherently dendritic in nature, and turbid water may not necessarily flow equally throughout an entire catchment. Tributaries have the potential to serve as refugia from temporarily turbid water in the main stem. As ayu (Plecoglossus altivelis) are one of the important species for the inland fisheries industry and recreational fishing, their behaviour in a branched river network is a primary concern. We released ayu with radio‐transmitting tags into two experimental streams that joined at a downstream pool. Turbid water was released into one of the streams, and we identified whether avoidance movement of ayu between the two experimental streams was triggered by flowing turbid water. The suspended solid concentration elevated rapidly after adding the turbid water and was maintained at more than 400 mg/L for 3 hr. Avoidance movement of ayu was triggered when the suspended solid concentration exceeded approximately 200 mg/L. Then, most ayu moved from the turbid stream to the nonturbid one, leading to a difference in ayu density between the two streams. Therefore, we demonstrated that turbid water induced interstream movement of a commercially important ayu as refuge behaviour, suggesting that river connectivity is important for the persistence of the ayu.     

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

Evaluating the rumen‐protected lysine stability in forage‐based total mixed rations in vitro and determining the lysine Brix value

Three rumen‐protected lysine (RPL) products (AjiPro^®‐L, LysiPEARL^TM, and Feedtech Bypass Lysine^TM: A, B, and C, respectively) were tested for stability in two forage‐based total mixed rations (TMR1, 41.3% dry matter (DM), and TMR2, 49.5% DM) (experiment 1) and for Brix value (experiment 2). In experiment 1, each RPL product (2 g each) and TMR diet (200 g) were mixed and stored in plastic bags at 20°C for 0, 1, 3, 6, 12, 24, and 48 hr. In experiment 2, each RPL product (2 g) was dispensed into ion‐exchanged water (20 ml) and kept at 20°C for 0, 0.5, 1, 3, 6, 12, 24, and 48 hr. At each time point, free lysine (Lys) content and Brix values of extracts were measured, and Lys release (LR, %) was calculated. All RPL products LR% varied with varying diets DM and increased with increasing of time exposed to diets; it was highest in C, followed by B, and then A. Water LR% positively correlated with that from diets and with Brix values of Lys dissociated in water. Our results indicated that Lys dissociation from RPL products is affected by diet DM content. Brix value may be used as a potential marker for RPL protection efficacy.     

Circadian variations in salivary chromogranin a concentrations during a 24-hour period in dogs

The purpose of this study was to determine if salivary chromogranin a secretion in dogs exhibits a circadian rhythm. Saliva sampling was performed during three different sessions occurring in three nonconsecutive 24-h periods. Sixteen healthy adult beagle dogs (8 males and 8 females) were moved to a sampling room and housed individually in cages. Saliva samples were obtained every 4 h from 12:00 p.m. to 12:00 p.m. the following day. In the interest of habituation, saliva was obtained hourly from each dog 3 h before the experiment was started. Salivary chromogranin A concentrations were measured using an enzyme-linked immunosorbent assay. No circadian rhythm was detected for salivary chromogranin A secretion, and no differences in salivary chromogranin A concentrations measured every 4 h were demonstrated during the 24-h cycle in dogs.     

How to determine the appropriate mortality in experimental larval rearing?

Survival in larval rearing experiments is difficult to estimate due to accidental losses and periodic sampling. The number of sampled fish can be a large proportion of the stocked ones, making it difficult to calculate the overall survival rate and mortality coefficient as this is based on the initial number. Here, a new method of calculating survival is proposed using the mortality coefficient. When the initial stocking density and sampled and final numbers are known, and assuming that mortality coefficient is constant, the final number of fishes can be represented by the formula N _t = e^−mt (N ₀ − ΣN _Sne ^mdn), where t is rearing period (days), N ₀ indicates initial number, N _t indicates the survival number at t days of rearing, m is the natural mortality coefficient, N _Sn is the sampled number in the nth sampling, and dn is the rearing period until removal of the nth sample. The provisional mortality coefficient is calculated from initial and final stocking numbers. Then values for the natural mortality coefficient are substituted into the formula with successive approximation. The coefficient, which most closely approximates the actual survival, is determined as the best fit natural mortality coefficient. Examples of larval experiments are provided to demonstrate the method and show that survival is often underestimated using traditional methods.     

Codend selectivity for jack mackerel and whitefin jack and unequal split parameter estimates observed in trouser trawl experiments

Codend selectivity for the jack mackerel Trachurus japonicus and the whitefin jack Kaiwarinus equula were evaluated based on data from trouser trawl experiments carried out in the East China Sea, using a test codend of 60 mm diamond mesh and a control codend made of minnow net with a square mesh of 9 mm bar length. Between-haul variations in parameters and the mean selection curves were tested with the catch data in the SELECT approach, and then the model of between-haul variation in the split parameter with the mean selection curve was chosen as the best fit using Akaike’s information criterion model selection. The 50% retention lengths and the selection ranges were 11.4 and 3.36 cm for jack mackerel and 8.83 and 0.93 cm for whitefin jack, respectively. The selection curve for whitefin jack was sharp, whereas that for jack mackerel was relatively wide. As the estimated split parameters indicated, about 80% of the whitefin jack entered the control codend, but 85 and 90% of the jack mackerel entered the control codend in the second and third hauls, respectively. The inequality in the split parameter is discussed from the viewpoint of the animal’s swimming behavior and water movement based on underwater video observations.