PCR Amplification of the Gene acmA Differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.     

Terrigenous sediment-dominated reef platform infilling: an unexpected precursor to reef island formation and a test of the reef platform size–island age model in the Pacific

Low-lying coral reef islands are considered highly vulnerable to climate change, necessitating an improved understanding of when and why they form, and how the timing of formation varies within and among regions. Several testable models have been proposed that explain inter-regional variability as a function of sea-level history and, more recently, a reef platform size model has been proposed from the Maldives (central Indian Ocean) to explain intra-regional (intra-atoll) variability. Here we present chronostratigraphic data from Pipon Island, northern Great Barrier Reef (GBR), enabling us to test the applicability of existing regional island evolution models, and the platform size control hypothesis in a Pacific context. We show that reef platform infilling occurred rapidly (~4–5 mm yr⁻¹) under a “bucket-fill” type scenario. Unusually, this infilling was dominated by terrigenous sedimentation, with platform filling and subsequent reef flat formation complete by ~5000 calibrated years BP (cal BP). Reef flat exposure as sea levels slowly fell post highstand facilitated a shift towards intertidal and subaerial-dominated sedimentation. Our data suggest, however, a lag of ~1500 yr before island initiation (at ~3200 cal BP), i.e. later than that reported from smaller and more evolutionarily mature reef platforms in the region. Our data thus support: (1) the hypothesis that platform size acts to influence the timing of platform filling and subsequent island development at intra-regional scales; and (2) the hypothesis that the low wooded islands of the northern GBR conform to a model of island formation above an elevated reef flat under falling sea levels.

     

Mechanistic studies of displacer–protein binding in chemically selective displacement systems using NMR and MD simulations

A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair α‐chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1‐(4‐chlorobenzyl)piperidin‐3‐aminesulfate and N′1′‐(4‐methyl‐quinolin‐2‐yl)‐ethane‐1,2‐diamine dinitrate, and one non‐selective displacer, spermidine, were selected as model systems to investigate the mechanism of chemically selective displacement chromatography. Saturation transfer difference (STD) NMR was used to directly evaluate displacer–protein binding. The results indicated that while binding occurred between the two chemically selective displacers and the more hydrophobic protein, α‐chymotrypsin, no binding was observed with ribonuclease A. Further, the non‐selective displacer, spermidine, was not observed to bind to either protein. Importantly, the binding event was observed to occur primarily on the aromatic portion of the selective displacers. Extensive molecular dynamic simulations of protein–displacer–water solution were also carried out. The MD results corroborated the NMR findings demonstrating that the binding of selective displacers occurred primarily on hydrophobic surface patches of α‐chymotrypsin, while no significant long term binding to ribonuclease A was observed. The non‐selective displacer did not show significant binding to either of the proteins. MD simulations also indicated that the charged amine group of the selective displacers in the bound state was primarily oriented towards the solvent, potentially facilitating their interaction with a resin surface. These results directly confirm that selective binding between a protein and displacer is the mechanism by which chemically selective displacement occurs. This opens up many possibilities for future molecular design of selective displacers for a range of applications. Biotechnol. Bioeng. 2009;102: 1428–1437. © 2008 Wiley Periodicals, Inc.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation

We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1+1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160×10⁶ mL⁻¹ and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl^® (20% egg yolk) and UL extender (Tes-Tris-fructose, 15% egg yolk, 4% glycerol). Triladyl^® yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl^®, Andromed^®, Bioxcell^®, and UL with 8% LDL (low-density lipoproteins). Triladyl^® and Andromed^® performed better than Bioxcell^® on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1+1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl^® and Andromed^® were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing.     

Influence of the Temperature and the Genotype of the HSP90AA1 Gene over Sperm Chromatin Stability in Manchega Rams

The present study addresses the effect of heat stress on males'' reproduction ability. For that, we have evaluated the sperm DNA fragmentation (DFI) by SCSA of ejaculates incubated at 37°C during 0, 24 and 48 hours after its collection, as a way to mimic the temperature circumstances to which spermatozoa will be subject to in the ewe uterus. The effects of temperature and temperature-humidity index (THI) from day 60 prior collection to the date of semen collection on DFI were examined. To better understand the causes determining the sensitivity of spermatozoa to heat, this study was conducted in 60 males with alternative genotypes for the SNP G/C₋₆₆₀ of the HSP90AA1 promoter, which encode for the Hsp90α protein. The Hsp90α protein predominates in the brain and testis, and its role in spermatogenesis has been described in several species. Ridge regression analyses showed that days 29 to 35 and 7 to 14 before sperm collection (bsc) were the most critical regarding the effect of heat stress over DFI values. Mixed model analyses revealed that DFI increases over a threshold of 30°C for maximum temperature and 22 for THI at days 29 to 35 and 7 to 14 bsc only in animals carrying the GG₋₆₆₀ genotype. The period 29–35 bsc coincide with the meiosis I process for which the effect of the Hsp90α has been described in mice. The period 7–14 bsc may correspond with later stages of the meiosis II and early stages of epididymal maturation in which the replacement of histones by protamines occurs. Because of GG₋₆₆₀ genotype has been associated to lower levels of HSP90AA1 expression, suboptimal amounts of HSP90AA1 mRNA in GG₋₆₆₀ animals under heat stress conditions make spermatozoa DNA more susceptible to be fragmented. Thus, selecting against the GG₋₆₆₀ genotype could decrease the DNA fragmentation and spermatozoa thermal susceptibility in the heat season, and its putative subsequent fertility gains.     

A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation

Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage.     

DNA probes for detection of copper resistance genes in Xanthomonas campestris pv. vesicatoria.

The copper resistance (Cur) genes encoded on pXV10A, a 190-kb plasmid in Xanthomonas campestris pv. vesicatoria XV10, were isolated on a 44-kb cosmid clone designated pCuR1. Tn5 mutagenesis of pCuR1 indicated that a 4.0-kb region was required for copper resistance. Three restriction fragments located within the 4.0-kb region demonstrated high specificity for the Cur genes present in X. campestris pv. vesicatoria and will be useful in monitoring the presence of these genes in the environment.