Temperature is the evil twin: effects of increased temperature and ocean acidification on reproduction in a reef fish.

Reproduction in many organisms can be disrupted by changes to the physical environment, such as those predicted to occur during climate change. Marine organisms face the dual climate change threats of increasing temperature and ocean acidification, yet no studies have examined the potential interactive effects of these stressors on reproduction in marine fishes. We used a long-term experiment to test the interactive effects of increased temperature and CO2 on the reproductive performance of the anemonefish, Amphiprion melanopus. Adult breeding pairs were kept for 10 months at three temperatures (28.5°C [+0.0°C], 30.0°C [-1.5°C] and 31.5°C [+3.0°C]) cross-factored with three CO2 levels (a current-day control [417 µatm] and moderate [644 µatm] and high [1134 µatm]) treatments consistent with the range of CO2 projections for the year 2100. We recorded each egg clutch produced during the breeding season, the number of eggs laid per clutch, average egg size, fertilization success, survival to hatching, hatchling length, and yolk provisioning. Adult body condition, hepatosomatic index, gonadosomatic index, and plasma 17ß-estradiol concentrations were measured at the end of the breeding season to determine the effect of prolonged exposure to increased temperature and elevated. CO2 on adults, and to examine potential physiological mechanisms for changes in reproduction. Temperature had by far the stronger influence on reproduction, with clear declines in reproduction occurring in the +1.5°C treatment and ceasing altogether in the +3.0°C treatment. In contrast, CO2 had a minimal effect on the majority of reproductive traits measured, but caused a decline in offspring quality in combination with elevated temperature. We detected no significant effect of temperature or Co2 on adult body condition or hepatosomatic index. Elevated temperature had a significant negative effect on plasma 17ß-estradiol concentrations, suggesting that declines in reproduction with increasing temperature were due to the thermal sensitivity of reproductive hormones rather than a reduction in energy available for reproduction. Our results show that elevated temperature exerts a stronger influence than high CO2 on reproduction in A. melanopus. Understanding how these two environmental variables interact to affect the reproductive performance of marine organisms will be important for predicting the future impacts of climate change.

Rapid assessment of postural instability in Parkinson's disease (RAPID): a pilot study.

BACKGROUND: The Fahn's pull (or retropulsion) test is an item in the motor section of the Unified Parkinson's Disease Rating Scale, which is used almost exclusively to classify postural instability in Parkinson's disease (PD). However, the test is hard to standardize and is often performed incorrectly, making it hard to interpret. Moreover, it may not be safe to administer in patients who experience pain in the shoulders, neck, trunk and/or lower extremities. Identifying and grading postural instability in PD without requiring a physical challenge would not only be useful for the clinician but would assist patients and caregivers in its recognition. We propose the use of the rapid assessment of postural instability in Parkinson's disease (RAPID) questionnaire as a non-physical assessment tool. METHODS: We determined the associations between the pull test and items on a risk-assessment questionnaire that consisted of three parts: activities of daily living, fear of falling, and frequency of falling. RESULTS: Significant correlations were found between the pull test and the predictor variables, which ranged between 0.51 and 0.56 whilst the correlations amongst the predictor variables ranged between 0.58 and 0.70. The three parts of the questionnaire, when used in combination, produced a 96% sensitivity in the classification of postural instability. CONCLUSIONS: The RAPID questionnaire can be used as an adjunct to the pull test or solely if the pull test is contraindicated. It may also be possible to administer the questionnaire via the telephone or Internet. It is hoped that the rapid identification of postural instability would lead to fewer falls.

TPH2 5'- and 3'-regulatory polymorphisms are differentially associated with HPA axis function and self-injurious behavior in rhesus monkeys.

Tryptophan hydroxylase-2 (TPH2) synthesizes neuronal serotonin and is linked to numerous behavioral traits. We have previously characterized the functionality of polymorphisms (especially 2051A>C) in 3'-untranslated region (3'-UTR) of rhesus monkey TPH2 (rhTPH2). This study further assessed the functionality of additional polymorphisms (-1605T>C, -1491Tn, -1485(AT)n, -1454A>G, -1325In>Del and -363T>G) in rhTPH2 5'-flanking region (5'-FR), and evaluated the effects of rhTPH2 5' and 3' genotypes on central serotonin turnover, hypothalamic-pituitary-adrenal (HPA) axis function and self-injurious behavior (SIB) in 32 unrelated adult male monkeys of Indian origin. Haplotypes of the rhTPH2 5'-FR polymorphisms exert a significant, cell-dependent effect on reporter gene expression, primarily conferred by -1485(AT)n. The -1485(AT)n and 2051A>C polymorphisms interact to influence cerebrospinal fluid (CSF) 5-HIAA and plasma adrenocorticotropic hormone (ACTH) in the afternoon. While -1485(AT)n exerts significant main effects on the afternoon cortisol level and nocturnal HPA negative feedback, 2051A>C has significant main effects on the morning cortisol level and cortisol response to ACTH challenge, as well as marginally significant main effects on the daytime HPA negative feedback and self-biting rate. In addition, the genotype/allele frequency of the 5'-FR -1325Ins>Del differed significantly between the self-wounders and non-wounders, whereas 3'-UTR 2128S>L polymorphism differed significantly in genotype/allele frequency between the high- and low-frequency biters. This study shows the functionality of rhTPH2 5'-FR polymorphisms, and provides evidence for the differential association of rhTPH2 5'-FR and 3'-UTR polymorphisms with HPA axis function and SIB. Our findings shed light on the role of TPH2 gene variance in physiology and behavioral traits, and also contribute to the understanding of the pathophysiology and genetics of SIB.

Resources for genetic management and genomics research on non-human primates at the National Primate Research Centers (NPRCs).

The National Primate Research Centers (NPRCs) established Working Groups (WGs) for developing resources and mechanisms to facilitate collaborations among non-human primate (NHP) researchers. Here we report the progress of the Genome Banking and the Genetics and Genomics WGs in developing resources to advance the exchange, analysis and comparison of NHP genetic and genomic data across the NPRCs. The Genome Banking WG has established a National NHP DNA bank comprising 1250 DNA samples from unrelated animals and family trios from the 10 NHP species housed within the NPRC system. The Genetics and Genomics WG is developing SNP arrays that will provide a uniform, highly informative, efficient and low-cost method for rhesus and long-tailed macaque genotyping across the eight NPRCs. This WG is also establishing a Biomedical Informatics Research Network-based portal for shared bioinformatics resources including vital statistics, genotype and population data and information on the National NHP DNA bank.

Rhesus monkey tryptophan hydroxylase-2 coding region haplotypes affect mRNA stability.

Tryptophan hydroxylase-2 (TPH2) synthesizes neuronal 5-HT and its genetic variance is associated with numerous behavioral traits and psychiatric disorders. This study characterized the functional significance of two nonsynonymous single nucleotide polymorphisms (SNPs) (C74A and G223A) in rhesus monkey TPH2 (mTPH2). Four haplotypes of mTPH2 were cloned into pcDNA3.1 and stably transfected into PC12 cells. The levels of mTPH2 mRNA and protein were assessed by quantitative real-time PCR and Western blot, respectively, while the intracellular 5-HT was measured by enzyme-linked immunosorbent assay (ELISA). The variant A-A haplotype showed significantly higher levels of mTPH2 mRNA and protein, as well as significantly higher 5-HT production than the wild-type C-G haplotype, while the other two variant haplotypes (C-A and A-G) also tended to produce more 5-HT than C-G haplotype when stably expressed in PC12 cells. Both C74A and G223A were predicted to change mRNA secondary structure, and analysis of the mRNA stability showed that the wild-type C-G haplotype mRNA degrades more quickly than mRNAs of the mutant mTPH2 haplotypes in both stable PC12 and transient HEK-293 cells. This study demonstrates that nonsynonymous SNPs in mTPH2 can affect mRNA stability. Our findings provide an additional mechanism by which nonsynonymous SNPs affect TPH2 function, and further our understanding of TPH2 gene expression regulation.

Functional variation in the 3' untranslated region of the serotonin transporter in human and rhesus macaque.

The serotonin system underlies a wide variety of behavioral traits and its dysregulation is the cause of numerous neuropsychiatric disorders. Among genes involved in the system, the serotonin transporter (SERT) is integral and has been repeatedly shown to be associated with disease as well as being a primary drug target. In addition to promoter region variation, we identify here variation in a regulatory region in the 3' untranslated region (UTR) of the SERT gene in both humans and rhesus macaques. We comprehensively survey the 3' UTR of SLC6A4 in Indian-origin rhesus macaques to identify three single nucleotide polymorphisms (SNPs) creating two haplotypes, both derived from an ancestral sequence, that represent the vast majority of the alleles in the population. Through the use of a luciferase reporter gene assay, we are able to show that not only do these alleles have differential effects on gene expression, modulated through changes in messenger RNA stability, but that different commonly occurring SNPs in the human 3' UTR also have similar effects. This finding not only offers additional insight into the regulation, and thus dysregulation, of SERT expression, but also suggests the role of natural selection in maintaining both high and low SERT expression levels broadly across populations of multiple primate species.

Increased signal in the subarachnoid space on fluid-attenuated inversion recovery imaging associated with the clearance dynamics of gadolinium chelate: a potential diagnostic pitfall.

BACKGROUND AND PURPOSE: Hyperintense CSF in the subarachnoid space (SAS) on fluid-attenuated inversion recovery (FLAIR) imaging has been reported in numerous pathologic conditions, including subarachnoid hemorrhage, meningitis, meningeal carcinomatosis, superior sagittal thrombosis, adjacent tumors, status epilepticus, and stroke. It has also been reported in otherwise healthy patients undergoing anesthesia with supplemental oxygen. We present a series of 11 patients with hyperintense CSF signal intensity in the SAS on FLAIR imaging after previous administration of gadolinium chelate. MATERIALS AND METHODS: Head MR images of patients who had a prior gadolinium-enhanced body, spine, or brain MR imaging and who had increased signal intensity in the SAS on FLAIR images were prospectively and retrospectively reviewed. Correlation was made with the clinical and laboratory findings. RESULTS: Eight of the 11 patients had negative findings on lumbar punctures. Seven patients had either chronic renal insufficiency or acute renal failure, but the remaining 4 had normal renal function. Nine patients had no other significant intracranial abnormalities, and 2 patients had acute infarcts remote from the CSF hyperintensity. One patient had follow-up studies at 24 and 48 hours, documenting resolution of the CSF hyperintensities. CONCLUSION: Given the sharp rise in volume of contrast-enhanced MR imaging studies, it is inevitable that some patients will have undergone a contrast-enhanced MR imaging 24-48 hours before an MR imaging of the brain. The neuroradiologist should be aware that previous administration of gadolinium chelate can cause increased signal intensity in the SAS on FLAIR imaging in patients with or without a history of renal insufficiency and without abnormalities known to disrupt the blood-brain barrier.

Tryptophan hydroxylase-2 gene polymorphisms in rhesus monkeys: association with hypothalamic-pituitary-adrenal axis function and in vitro gene expression.

Tryptophan hydroxylase-2 (TPH2) is a newly identified second form of TPH responsible for serotonin synthesis in the brain and has been increasingly implicated as a contributor to the etiology of various psychiatric disorders. In this study, we have identified the constellation of polymorphisms in rhesus monkey TPH2 and investigated genotype/phenotype association as well as gene expression effects of specific polymorphisms. Genomic DNA was obtained from 247 rhesus monkeys, among which 24 had been previously examined for plasma cortisol level, dexamethasone suppression, and combined dexmethasone/ACTH challenge. Polymorphisms in all exons, splicing junctions and approximately 2 kb of the 5'-flanking region (5'-FR) of TPH2 were identified by sequencing. We identified 17 single nucleotide polymorphisms (SNPs) including two that are predictive of amino-acid change (25Pro>His and 75Gly>Ser, respectively), two mononucleotide repeats, one dinucleotide repeat, and one 159-bp insertion polymorphism. The 3'-UTR polymorphisms were significantly associated with hypothalamic-pituitary-adrenal (HPA) axis activity, especially 2051A>C, which was strikingly correlated with plasma cortisol level in the morning only (F=10.203, P=0.001). Luciferase reporter gene assays showed that the 3'-UTR polymorphisms and haplotypes had a profound effect on in vitro gene expression. Accordingly, these investigations revealed that polymorphisms in 3'-UTR of rhesus monkey TPH2 modulate HPA axis function, presumably by affecting levels of TPH2 expression.

Superficial siderosis.

BACKGROUND: Superficial siderosis (SS) of the CNS is caused by repeated slow hemorrhage into the subarachnoid space with resultant hemosiderin deposition in the subpial layers of the brain and spinal cord. Despite extensive investigations, the cause of bleeding is frequently undetermined. OBJECTIVES: To review the clinical and imaging features of 30 consecutive patients with SS and provide insights into the underlying causes of subarachnoid bleeding in this disabling disorder. METHODS: The authors reviewed the medical records of 30 consecutive patients with clinical and MRI evidence of SS. RESULTS: The commonest neurologic manifestations included gait ataxia and hearing impairment. A clinical history of subarachnoid hemorrhage was relatively rare. Possible predisposing conditions were identified on history in 22 patients, the commonest being a prior trauma (15 patients). In addition to the characteristic MRI findings of SS, 18 patients had abnormalities on MRI possibly related to chronic bleeding. The most common of these was the presence of a fluid-filled collection in the spinal canal seen in 14 patients. CONCLUSIONS: A history of prior subarachnoid hemorrhage is often absent in patients with superficial siderosis (SS). A past history of trauma is common. Prior intradural surgery may be an additional risk factor. Xanthochromia or the presence of red blood cells in the CSF is a common finding. Only rarely does angiography demonstrate the bleeding source. The presence of a fluid-filled collection in the spinal canal is a common finding on MRI and is likely related to the SS. With longitudinally extensive cavities, a dynamic CT myelogram may help localize the defect and direct the site of laminectomy. Surgical repair of a dural defect, if present, should be considered. Surgical correction of bleeding should be documented by CSF examination months after surgery. Friable vessels in the dural defect are a possible source of the chronic bleeding.

Effect of housing system and boar exposure on estrus expression in weaned sows.

Reproductive efficiency depends on detection of estrus, which may be influenced by housing and boar exposure. This experiment investigated the effects of housing system and boar contact on measures of estrus in weaned sows. Mixed-parity sows were randomly assigned to be weaned into gestation crates away from boars (AWC, n = 45), into pens away from boars (AWP, n = 42), or into pens adjacent to a mature boar (ADJ, n = 46). Estrus detection was initiated at approximately 0700 (0 h) and again at 0.25-, 0.5-, 1-, 2-, 4-, and 8-h intervals beginning on d 4 and continuing through d 7 following weaning. Estrus detection involved observation of the standing response after application of nose-to-nose boar exposure, backpressure, and side rubbing. For the AWC sows, a mature boar was moved to the front of the crates for a 10-min period and then removed. Sows housed in AWP were moved approximately 15 m to an empty pen adjacent to a mature boar for a 10-min period, and then returned to their pen. Sows housed ADJ were not moved and estrus detection was performed in their home pen for a 10-min period. The proportion of sows expressing estrus within 7 d from weaning was lowest for ADJ (80%, 37/46) compared with AWP (98%, 41/42) and AWC (96%, 43/45; P < 0.05). There was an effect of interval from weaning to estrus on the percentage of sows expressing estrus, but there was no interaction with treatment. Sows in AWC and AWP (4.7 d) had decreased (P = 0.01) intervals from weaning to estrus compared with ADJ (5.2 d). The duration of estrus was also shorter (P < 0.001) for ADJ (45 h) compared with AWC (58 h) or AWP (62 h). There was a treatment x interval x day of estrus effect for the percentage of sows expressing estrus. After detection of the first standing response on the first day of estrus, only 62 to 82% of sows were detected standing over the next 2 h for all treatments. However, at 4 to 8 h, this increased to 85 to 98% for the AWC and AWP sows, but <73% of the ADJ sows were detected during this period. On the second day of estrus, estrus expression was not influenced by interval for the AWC and AWP sows and was between 90 to 100% during the 8-h period, whereas ADJ sow detection rates were between 68 to 88%. These data suggest that housing sows adjacent to boars negatively affects estrus expression and detection. In addition, refractory behavior occurs in approximately 30 to 40% of sows and is influenced by housing relative to the boar, day of estrus, and interval from last boar exposure.

A mu-opioid receptor single nucleotide polymorphism in rhesus monkey: association with stress response and aggression.

Variations in the human mu-opioid receptor gene have driven exploration of their biochemical, physiological and pathological relevance. We investigated the existence of variations in the nonhuman primate mu-opioid receptor gene to determine whether nonhuman primates can model genotype/phenotype associations of relevance to humans. Similar to the A118G single nucleotide polymorphism (SNP) in the human mu-opioid receptor gene, a SNP discovered in the rhesus monkey mu-opioid receptor gene (C77G) alters an amino acid in the N-terminal arm of the receptor (arginine for proline at position 26). Two mu-opioid receptor coding regions isolated from a single heterozygous (C77/G77) rhesus monkey brain were expressed in HEK-293 cells and characterized in radioreceptor assays. Paralleling the findings of increased affinity of beta-endorphin by the A118G allele in the human, the rhesus monkey mu-opioid receptor protein derived from the G77-containing clone demonstrated a 3.5-fold greater affinity for beta-endorphin than the receptor derived from the C77-containing clone. An assay developed to assess the incidence of the C77G SNP in a behaviorally and physiologically characterized cohort of rhesus monkeys (n=32) indicated that 44% were homozygous for C77-containing alleles, 50% were heterozygous and 6% were homozygous for G77-containing alleles. The presence of G77-containing alleles was associated with significantly lower basal and ACTH-stimulated plasma cortisol levels (P<0.03-0.05 and P<0.02, respectively) and a significantly higher aggressive threat score (P<0.05) in vivo. In a cohort of 20 monkeys, a trend towards an inverse correlation between aggressive threat and plasma cortisol levels was observed. The findings suggest that mu-opioid receptor haplotypes in monkeys can contribute to individual variability in stress response and related aggression. The data support the use of nonhuman primates to investigate mu-opioid receptor genotype/phenotype relations of relevance to humans.

Pseudosubarachnoid haemorrhage in subdural haematoma.

Two patients with large bilateral subdural haematomas with patterns of non-enhanced brain computed tomography (CT) falsely suggesting coexistent subarachnoid haemorrhage are presented. The CT images showed marked effacement of the basal cisterns with hyperdense signal along the tentorium, sylvian fissure, and the perimesencephalic cisterns. In both cases, the suspicion of subarachnoid haemorrhage led to the performance of angiographic studies to rule out vascular lesions. Thus, recognition of this radiological feature is important to avoid unnecessary testing and treatment delay.

Influence of hormone supplementation to extended semen on artificial insemination, uterine contractions, establishment of a sperm reservoir, and fertility in swine.

This study was performed to quantify the effect of hormone addition to semen using a low-fertility model to evaluate its effectiveness and mode of action. At 24 h after the onset of estrus, all gilts received a single low-dose AI (0.5 x 10(9) sperm/80 mL) with no hormone (control, C), estrogens (E, 11.5 microg), PGF2alpha (PG, 5 mg of Lutalyse), or oxytocin (OT, 4 IU), which were then evaluated for semen backflow (n = 48), oviductal and uterine sperm numbers (n = 28), uterine contractions (n = 12), pregnancy rate (PR, n = 120), and number of fetuses (n = 67). In Exp. 1, backflow of semen from the uterus was collected for 8 h after AI, whereas PR and fetuses were assessed at d 25 to 30 after AI. In Exp. 2, backflow was collected and reproductive tracts flushed to determine sperm numbers in the oviducts and the anterior segments of the uterus. In Exp. 3, sows were monitored for uterine contractions for 1 h before AI and for 2 h after AI. In Exp. 1, there was a treatment x time interaction for fluid loss (P < 0.001), but by 8 h after AI, there was no difference in the total volume (70 +/- 1 mL) of semen lost between hormone treatments (85%) compared to controls (90%). There was also a treatment x time interaction (P < 0.05) for number of sperm lost in the backflow (2.1 +/- 0.1 x 10(8)), but by 8 h following AI, there was no effect on total sperm lost for the hormone treatments (38%) compared to C (54%). There was a trend (P = 0.10) for increased numbers of sperm in the uteri of hormone-treated gilts (6.0 +/- 1.3 x 10(4)) compared with C gilts (2.2 +/- 1.3 x 10(4), but there was no effect of treatment on sperm numbers in the oviducts (3.2 +/- 1.3 x 10(4)). Within 0.5 h of AI, there was an increase in the frequency of contractions for PG compared with the other treatments (14.2 vs. 6.3/h, P < 0.005), however there was no effect on amplitude (54 mmHg) or duration (35 s) of contractions. The PR was not influenced by treatment and averaged 54% (P > 0.60), but total numbers of healthy fetuses were increased (P < 0.04) by PG (8.7) and tended (P = 0.06) to be increased for OT (8.4), but not for E (7.2) compared to C (5.8). Hormone addition to semen increased numbers of fetuses and this may be related to an alteration in the pattern of fluid and sperm loss after AI and a tendency for increased numbers of sperm in the anterior segment of the uterus. Therefore, in situations of lowered fertility, hormone addition could be a strategy to limit infertility in swine.

Effect of boar exposure at time of insemination on factors influencing fertility in gilts.

The effect of boar exposure during artificial insemination (AI) on semen backflow, fertilization, and embryo quality was evaluated. Gilts (approximately 170 d) were induced into estrus with PG600, and ovulation was synchronized using hCG 72 h later. Estrus detection was initiated after PG600 and continued at 12-h intervals. At estrus, gilts were allotted to receive boar exposure (BE, n = 20) or no boar exposure (NBE, n = 20) during AI. Gilts receiving NBE were identified to be in estrus prior to AI and the boar was then removed for 1 h, whereas gilts in the BE group received 15 min of exposure during AI. Insemination occurred in crates at 12 and 24 h after onset of estrus with 3 x 10(9) sperm/80 mL. Backflow was collected continuously with samples taken at time 0, (during AI), and at 0.25, 0.5, 0.75, 1, 2, 4, and 8 h after first and second AI. The effect of treatment was evaluated for time of insemination (min), backflow (mL), and sperm in backflow samples. Oviducts were flushed 2 d after first AI to evaluate the effect oftreatment on fertilization rate, accessory sperm numbers on embryos (scored 1 to 5), and embryo quality. There was no effect of first or second AI; therefore, data were pooled. Average duration of AI was 3.7 +/- 0.2 min and was not influenced by BE (P < 0.10). However, during the initial stage of AI, BE reduced the volume of semen (18.6 vs 32.4 +/- 3 mL) and the number of sperm lost (0.8 vs 1.3 +/- 0.15 x 10(9) sperm) compared to NBE (P < 0.05). There was a treatment x time effect (P < 0.05) for volume of backflow. By 45 min, the BE gilts lost more volume (9.0 vs 3.6 mL) compared to the NBE group, but sperm loss did not differ. Between 1 and 8 h after AI, neither volume nor sperm loss was influenced by treatment. By 8 h, total leakage (65 vs 63 mL) and total sperm loss (1.6 x 10(9) vs 1.8 x 10(9) sperm) were not influenced by BE (P > 0.10). However, more accessory sperm (P < 0.01) were found on embryos for the NBE (> or = 11 sperm/embryo) compared to BE embryos (< or = 10 sperm/embryo). Despite this observation, percentages of fertilized embryos (99.5 +/- 0.5 %) and number of embryos (11.5 +/- 0.1) were not different (P > 0.10). In conclusion, AI in the presence of a mature boar did not affect total semen leakage, sperm loss, fertilized embryos, or embryo quality. The importance of boar exposure during insemination was evident from less leakage during insemination, but had no effect on fertility; this suggests that the elimination of boar exposure during AI may not be deleterious to reproductive performance.

Effect of frequency of boar exposure and adjusted mating times on measures of reproductive performance in weaned sows.

In weaned sows, reduced reproductive performance can result from failure or delayed return to estrus or improper timing of insemination. This experiment evaluated the effect of increased frequency of boar exposure and adjusted mating times on reproductive performance. Sows of mixed parity were weaned approximately 18.7 d after parturition and allotted by genotype, parity, and lactation length to boar exposure frequency of once daily (1X, n = 66), twice daily every 12 h (2X, n = 61), or three times daily exposure at 8-h intervals (3X, n = 60). Sows were weaned into crates and boar exposure was initiated 3 d after weaning. Once estrus was detected, ultrasound was performed every 8 h to determine time of ovulation. All sows were artificially inseminated twice in the 1X group at 0 and 24 h, in the 2X group at 12 and 24 h, and in the 3X group at 16 and 32 h after onset of estrus. The weaning-to-estrus interval was not influenced by treatment and averaged 4.5 d. The percentage of sows expressing estrus in 8 d was higher (P < 0.05) for 1X (97.3%) compared with 2X (92.8%) but not the 3X group (94.0%). The percentage of sows ovulating after estrus was not influenced by treatment (P > 0.10) and averaged 96.5%. Estrus-to-ovulation interval was not affected by treatment (44.7 h) but was influenced by weaning-to-estrus interval (P < 0.0001). Length of estrus was influenced by treatment (P < 0.001), with estrus in the 1X (46.6 h) shorter than in the 2X (60.0 h) and 3X (67.0 h) treatments, and also by weaning-to-estrus interval (P < 0.001). The percentage of first inseminations occurring within 24 h before ovulation was increased (P < 0.001) in the 2X (62%) and 3X (66%) groups compared with the 1X group (28%) and was also influenced by parity (P < 0.001) and weaning-to-estrus interval (P < 0.05). The percentage of second services within 24 h before ovulation was not increased by any factor and averaged 78%. Farrowing rates were not increased (P > 0.10) for 2X (87.2%) and 3X (83.1%) treatments compared with 1X (75.0%). Total pigs born was also not affected by treatment, although 2X (11.2) and 3X (10.7) numbers were greater than 1X (10.0). It appears that once-daily estrus detection combined with delayed mating could achieve optimal reproductive performance.

Polymorphisms in the 3'-untranslated region of human and monkey dopamine transporter genes affect reporter gene expression.

Dopamine transporter (DAT) levels vary in normal subjects and deviate from the normal range in pathological states. We investigated mechanisms by which the DAT gene may influence DAT protein expression. As the 3'-untranslated region (3'-UTR) of the DAT gene varies with regard to length and single nucleotide polymorphisms (SNPs), we addressed whether the 3'-UTR of sequence-defined DAT alleles can differentially affect the level of reporter gene expression in vitro. We first established that within individual rhesus monkeys, two alleles of the DAT gene were expressed in the substantia nigra. We then transfected HEK-293 cells with HSV-TK- and SV40-driven luciferase expression vectors harboring downstream DAT 3'-UTR segments of alleles containing polymorphisms of length (human: 9 or 10 repeat units) or SNPs within alleles of fixed length (human: DraI-sensitive (DraI+) vs. DraI-insensitive (DraI-) 10-repeat alleles; rhesus monkey: Bst1107I-sensitive (Bst+) vs. Bst1107I-insensitive (Bst-) 12-repeat alleles). Vectors containing the 3'-UTR segment of a human DAT allele containing nine tandem repeat units resulted in significantly higher levels of luciferase production than analogous vectors containing 10 tandem repeat units. Depending on the promoter used, vectors containing the human or monkey 3'-UTR segments that differed on the basis of an SNP resulted in increases or decreases in luciferase gene expression. This report provides experimental evidence that variability in the length or the sequence of the 3'-UTR of the DAT gene may influence levels of DAT protein in the brain.

Liposome-encapsulated tumor necrosis factor-alpha enhances the effects of radiation against human colon tumor xenografts.

Recent reports have shown that tumor necrosis factor-alpha (TNF-alpha) can augment the effects of radiation against certain tumor types. However, the high concentrations of intravenous infusion of TNF-alpha needed to cause tumor regression can induce many systemic side effects. The aims of this study were to determine if TNF-alpha encapsulated in sterically stabilized (Stealth, ALZA Corporation, Mountain View, CA), PEGylated liposomes (SL) augments the antitumor effects of radiation and to compare its efficacy and possible toxicity with free TNF-alpha in the LS174T human colon tumor xenograft model. Nude mice were injected subcutaneously (s.c.) with LS174T cells and treated intravenously (i.v.) with Stealth-liposomal TNF-alpha (SL-TNF-alpha) with and without radiation or TNF-alpha with or without radiation when tumor size was approximately 200 mm(3). In phase 1, a significant decrease (p = 0.047) in tumor growth was observed with radiation at day 21 but not with SL-TNF-alpha or free TNF-alpha alone. By the end of phase 1 (day 27) with continued treatments, the SL-TNF-alpha plus radiation group had significantly smaller tumors (p = 0.044) than those in the free TNF-alpha plus radiation group. In phase 2, where a similar tumor growth reduction pattern was observed, the addition of TNF-alpha to radiation, either as free protein or within SL, increased lymphocyte activation and natural killer (NK) cell numbers in both blood and spleen. The effect was generally more pronounced with SL-TNF-alpha. Systemic toxicity, based on hematologic analyses and body weight, was absent or minimal. Collectively, the data show that pretreatment with SL-TNF-alpha can enhance more effectively, and possibly more safely, the effects of radiation against human colon tumor xenografts than can free TNF-alpha and that the increased antitumor action may involve upregulation of lymphocytes.

Non-amines, drugs without an amine nitrogen, potently block serotonin transport: novel antidepressant candidates?

The serotonin transporter (SERT) is a principal site of action of therapeutic antidepressants in the brain. Without exception, these inhibitors of serotonin transport contain an amine nitrogen in their structure. We previously demonstrated that novel compounds without an amine nitrogen in their structure (non-amines), blocked dopamine transport in cells transfected with the human dopamine transporter. The present study investigated whether, in the absence of an amine nitrogen, certain non-amines bind selectively to the SERT and block the transport of serotonin. At 10 microM concentration, select non-amines displayed no, or little, affinity for 9 serotonin, 5 dopamine, 7 adrenergic, 5 muscarinic cholinergic, 3 opiate and histamine receptors. The affinities of non-amines for [(3)H]citalopram binding sites on the SERT and their potencies for blocking [(3)H]serotonin transport were measured in cloned human SERT stably or transiently expressed in HEK-293. Whether oxa- or carba-based, non-amines bound to [(3)H]citalopram-labeled sites and blocked [(3)H]serotonin transport in the low nanomolar range, at values equal to or higher than those of some conventional antidepressants. A non-amine, O-1809, was 99-fold more selective for the serotonin over the dopamine transporter. As substituents on the aromatic ring of non-amines confer high affinity for the SERT, we investigated the hypothesis that aromatic-aromatic interactions may contribute significantly to non-amine/transporter association. A SERT mutant was produced in which a highly conserved aromatic amino acid, phenylalanine 548, was replaced by an alanine (F548A). Although the affinities of several non-amines were unchanged in the mutant SERT, the affinity of imipramine was decreased, revealing possible differences in amine and non-amine binding domains on the SERT. The similar affinities of non-amines and conventional antidepressant drugs for the SERT support the view that an amine nitrogen is not essential for drugs to block serotonin transport with high affinity. Non-amines open avenues for developing a new generation of antidepressants.