High-resolution protein structure determination by serial femtosecond crystallography

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.     

Expression of the μ Opioid Receptor and Effects of the Opioid Antagonist Naloxone on In Vitro Maturation of Oocytes Recovered from Anoestrous Bitches

The μ-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus–oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 × 10⁻⁶, 1 × 10⁻⁸ and 1 × 10⁻¹⁰  m . The rate of oocytes resuming meiosis after culture in presence of 1 × 10⁻⁶  m Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as δ and κ, and possible interactive effects of their antagonists on canine oocyte maturation.     

Second Oestrus Synchronization and Precocious Embryo Viability after Puberty Induction in Gilts by the Use of Gonadotrophin Treatment

The use of exogenous gonadotrophins in puberty inducement and ovulation synchronization is a technique that has a positive influence on the management of swine. The purpose of this study was to verify the effects of a second gonadotrophin treatment [equine chorionic gonadotrophin (eCG) and luteinizing hormone (LH), intramuscularly (i.m.)] upon the second oestrus synchronization and fertility in gilts. Seventy-one NAIMA (Pen Ar Lan) gilts had their first oestrus (puberty inducement) induced by a hormonal treatment (eCG and LH). Then, they were randomly distributed into two treatments, with (T1) and without (C) gonadotrophin treatment at the second oestrus. The animals were fed with a single ration (16% of crude protein and 3286.73 kcal ME/kg), and timed artificial insemination performed at the second oestrus. Gilts were slaughtered for embryo recovery and ovary examination about 5 days after insemination. There was no evidence of a difference in the percentage of the second oestrus expression (T1 - 90.90% and C - 86.84%), the duration of the oestrus cycle (T1 - 19.62 +/- 0.82 days and C - 19.67 +/- 4.14 days), the percentage of follicular cysts (T1 - 15.15% and C - 18.42%) and number of ovulations (T1 - 14.60 +/- 5.7 and C - 13.23 +/- 4.8) between treatments (p > 0.05). However, the hormonal treatment (T1) showed minor oestrus dispersion and embryo viability (T1 - 8.4 +/- 5.6 and C - 11.2 +/- 4.6) (p < 0.05). These results indicate that the better synchronization and expression of the second oestrus when using gonadotrophins (eCG and LH) is followed by a lower embryo viability, which is probably the consequence of the heterogeneous follicle recruitment during the injection of eCG.     

Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a cAMP analog

In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.     

Milk Progesterone Profile at and after Artificial Insemination in Repeat-Breeding Cows: Effects on Conception Rate and Embryonic Death

The aim of this study was to investigate whether the skim milk progesterone concentrations at artificial insemination (AI) and day of rise of post-ovulatory progesterone concentration thereafter affect the conception and embryonic death rates in repeat-breeding cows. Milk samples were obtained from 96 repeat-breeding cows that failed to conceive to three or more AIs. The samples were taken from the cows at the day of AI and three times/week until day 45 post-AI. Skim milk was obtained after centrifugation and used for progesterone assay. The cows with a progesterone concentration more than 0.5 ng/ml at AI showed a significantly higher incidence of late embryonic death than those having a progesterone concentration<0.5 ng/ml at AI (p<0.01). As the progesterone level at insemination rose, conception rate declined. A negative correlation was shown between conception rate and skim milk progesterone level at AI. Of 56 cows showing a rise of progesterone to 1 ng/ml or more within 6 days after AI, 28 cows (50%) conceived. On the contrary, only eight of 39 cows (20.5%) conceived when the progesterone rose up to 1 ng/ml after day 6 post-AI. We concluded that increased progesterone concentration at the time of AI and delayed rise of progesterone post-AI might lead to decrease in fertility in repeat-breeding cows.     

Pathological Conditions of the Reproductive Organs of Male Stray Dogs in the Tropics: Prevalence, Risk Factors, Morphological Findings and Testosterone Concentrations

The objective of this study was to estimate the prevalence of and risk factors for pathological conditions of the reproductive organs in stray dogs under tropical conditions. Three hundred and eighteen dogs were examined post-mortem in the period from 1 July 2002 to 30 June 2003. Before killing, a blood sample (from the cephalic vein) for testosterone assay was taken. Pathological conditions of the reproductive organs were found in 135 of the dogs (42.5%) and in 175 of the testes (64.8%). The most frequent pathologies found were testicular degeneration, cryptorchidism, testicular hypoplasia and testicular tumours (in 15.1%, 6.6%, 6.6% and 5.4% of the dogs and 15.1, 4.6, 6.0 and 3.5 of the testes, respectively). Transmissible venereal tumour (TVT) was seen in 5.4% of the dogs. Testicular degeneration was more common in old dogs and underweight dogs (p < 0.05). Testicular tumours were 14.3 times more common in cryptorchid dogs. Age was another important factor for the development of testicular tumours (p < 0.05). Lower levels of testosterone concentration (p < 0.05) were observed in dogs with advanced testicular degeneration (0.7 +/- 0.8 nM), dogs with hypoplastic testicles (0.8 +/- 0.9 nM) and dogs with one degenerated and one retained testis or with bilateral cryptorchidism (1.2 +/- 0.9 nM) compared to dogs with one or two normal testes (7.0 +/- 5.5 nM). Testicular volume and weight were significantly lower in degenerated, hypoplastic and retained testes compared with the contralateral normal testis. Some spermatogenic activity was found in three of the retained testes, producing oligozoospermic smears with a high percentage of sperm abnormalities. No comparable epidemiological data about male pathological conditions of the reproductive organs in the dog is available. The prevalence found in this study, yet, appears high.     

聚丙烯酰胺防治坡地水土流失田间试验研究

坡地篱用聚丙烯酰胺可以减少地表径流,提高土壤含水量,促进土壤沉降,增强土壤结构性和水稳性,因而提高了土壤的持水性和抗蚀性,减少了土壤侵蚀量和肥力流失量,有利于作物增产。施用聚丙烯酰胺可使产量增加１８．７％～３２．４％。施用聚丙烯酰胺对环境无不良影响,而且投入费用较低,是一条值得探索的防治坡地水土流失的新途径。     

History, instrumentation, and techniques of flexible endoscopic laser surgery in horses.

There are clearly a number of applications for which flexible endoscopic laser surgery has become the state of the art in equine surgery, and the Nd:YAG laser seems to be the most versatile instrument for this type of surgery. Nevertheless, it is critical to understand the advantages and disadvantages of each laser technique. For example, the Nd:YAG laser used in a noncontact fashion seems to be superior when ablation of tissue is required such as treatment of upper airway masses. Conversely, contact Nd:YAG laser techniques have proven themselves to be superior when more precise cutting is advantageous such as treatment of epiglottic entrapment. Ultimately, it seems that a range of lasers is necessary to ensure selection of the most appropriate technique, adding significantly to the expense of equipment but improving the outcome for a range of equine diseases.     

Standing Laparoscopic Peritoneal Flap Hernioplasty of the Vaginal Rings does not Modify the Sperm Production and Motility Characteristics in Intact Male Horses

Laparoscopic hernioplasty techniques have been developed in the recent years to avoid the recurrence of inguinal hernias and to spare the testicles for breeding purposes in stallions. However, there have been no previous comprehensive and systematic studies of the reproductive outcomes and prognoses for stallions after inguinal hernioplasty. Therefore, the objective of this study was to assess the possible effects of one of these techniques (standing laparoscopic peritoneal flap hernioplasty) on the sperm production and motility characteristics of six healthy stallions that received this procedure based on 1‐year follow‐ups. There were no significant differences in the measured sperm variables (assessments based on the DSO, MOT, PMOT, VSL, VCL and VAP) during 1‐year follow‐ups.