Intrastrain Differences Research Articles

Paralog transcriptional differentiation in the D. melanogaster-specific gene family Sdic across populations and spermatogenesis stages

How recently originated gene copies become stable genomic components remains uncertain as high sequence similarity of young duplicates precludes their functional characterization. The tandem multigene family Sdic is specific to Drosophila melanogaster and has been annotated across multiple reference-quality genome assemblies. Here we show the existence of a positive correlation between Sdic copy number and total expression, plus vast intrastrain differences in mRNA abundance among paralogs, using RNA-sequencing from testis of four strains with variable paralog composition. Single cell and nucleus RNA-sequencing data expose paralog expression differentiation in meiotic cell types within testis from third instar larva and adults. Additional RNA-sequencing across synthetic strains only differing in their Y chromosomes reveal a tissue-dependent trans-regulatory effect on Sdic: upregulation in testis and downregulation in male accessory gland. By leveraging paralog-specific expression information from tissue- and cell-specific data, our results elucidate the intraspecific functional diversification of a recently expanded tandem gene family.

Impaired beta-oxidation increases vulnerability to influenza A infection

Influenza A virus (IAV) infection casts a significant burden on society. It has particularly high morbidity and mortality rates in patients suffering from metabolic disorders. The aim of this study was to relate metabolic changes with IAV susceptibility using well-characterized inbred mouse models. We compared the highly susceptible DBA/2J (D2) mouse strain for which IAV infection is lethal with the C57BL/6J (B6) strain, which exhibits a moderate course of disease and survives IAV infection. Previous studies showed that D2 has higher insulin and glucose levels and is predisposed to develop diet-induced type 2 diabetes. Using high-resolution liquid chromatography–coupled MS, the plasma metabolomes of individual animals were repeatedly measured up to 30 days postinfection. The biggest metabolic difference between these strains in healthy and infected states was in the levels of malonylcarnitine, which was consistently increased 5-fold in D2. Other interstrain and intrastrain differences in healthy and infected animals were observed for acylcarnitines, glucose, branched-chain amino acids, and oxidized fatty acids. By mapping metabolic changes to canonical pathways, we found that mitochondrial beta-oxidation is likely disturbed in D2 animals. In noninfected D2 mice, this leads to increased glycerolipid production and reduced acylcarnitine production, whereas in infected D2 animals, peroxisomal beta-oxidation becomes strongly increased. From these studies, we conclude that metabolic changes caused by a distortion of mitochondrial and peroxisomal metabolism might impact the innate immune response in D2, leading to high viral titers and mortality.

Genomic Tools and Models for Investigating the Role of Germline Diversity in Mouse Antibody Repertoire Development

Abstract In mouse, the immunoglobulin (IG) loci span several Mb of the genome and contain hundreds of repeated, highly homologous sets of variable (V), diversity (D), and joining (J) genes that recombine in B cells to produce an individual’s expressed Ab repertoire. Similar to what has been reported in humans, recent data has shown significant levels of haplotype variation in the IG loci between commonly used inbred mouse strains, challenging the assumption that the IG loci are conserved across all strains of mice. We are using this intra-strain diversity to develop new models for studying the role of IG germline variation on Ab repertoire dynamics and function, questions that remain difficult to address in outbred human populations and pre-existing animal models. Given the diversity and complexity within the IG loci, the development of effective mouse models first requires the characterization of intra-strain differences and the construction of high-quality reference assemblies for the IG loci in several representative strains. To address this problem, we are using the Pacific Biosciences SMRT sequencing to sequence BAC clones spanning the IGH, IGK, and IGL loci in NOD/ShiLtJ and BALB/cByJ strains. We are also gaining additional insight on intra-strain diversity by profiling the expressed IGM, IGK, and IGL repertoires of 18 commonly used laboratory mouse strains. We have used this data to guide the construction of congenic lines on the C57BL/6 background, carrying divergent IGH or IGK loci from BALB/cByJ or NOD/ShiLtJ, respectively. Together, our data show significant germline diversity in our new IG assemblies, as well as divergent Ab repertoires in common lab mouse strains.

Magnetic resonance imaging reveals specific anatomical changes in the brain of Agat- and Gamt-mice attributed to creatine depletion and guanidinoacetate alteration.

Arginine:glycine amidinotransferase- and guanidinoacetate methyltransferase deficiency are severe neurodevelopmental disorders. It is not known whether mouse models of disease express a neuroanatomical phenotype. High-resolution magnetic resonance imaging (MRI) with advanced image analysis was performed in perfused, fixed mouse brains encapsulated with the skull from male, 10-12 week old Agat -exc and B6J.Cg-Gamt tm1Isb mice (n = 48; n = 8 per genotype, strain). T2-weighted MRI scans were nonlinearly aligned to a 3D atlas of the mouse brain with 62 structures identified. Local differences in brain shape related to genotype were assessed by analysis of deformation fields. Creatine (Cr) and guanidinoacetate (GAA) were measured with high-performance liquid chromatography (HPLC) in brain homogenates (n = 24; n = 4 per genotype, strain) after whole-body perfusion. Cr was decreased in the brain of Agat- and Gamt mutant mice. GAA was decreased in Agat-/- and increased in Gamt-/- . Body weight and brain volume were lower in Agat-/- than in Gamt-/- . The analysis of entire brain structures revealed corpus callosum, internal capsule, fimbria and hypothalamus being different between the genotypes in both strains. Eighteen and fourteen significant peaks (local areas of difference in relative size) were found in Agat- and Gamt mutants, respectively. Comparing Agat-/- with Gamt-/- , we found changes in three brain regions, lateral septum, amygdala, and medulla. Intra-strain differences in four brain structures can be associated with Cr deficiency, while the inter-strain differences in three brain structures of the mutant mice may relate to GAA. Correlating these neuroanatomical findings with gene expression data implies the role of Cr metabolism in the developing brain and the importance of early intervention in patients with Cr deficiency syndromes.

The relevance of inter- and intrastrain differences in mice and rats and their implications for models of seizures and epilepsy.

It is becoming increasingly clear that the genetic background of mice and rats, even in inbred strains, can have a profound influence on measures of seizure susceptibility and epilepsy. These differences can be capitalized upon through genetic mapping studies to reveal genes important for seizures and epilepsy. However, strain background and particularly mixed genetic backgrounds of transgenic animals need careful consideration in both the selection of strains and in the interpretation of results and conclusions. For instance, mice with targeted deletions of genes involved in epilepsy can have profoundly disparate phenotypes depending on the background strain. In this review, we discuss findings related to how this genetic heterogeneity has and can be utilized in the epilepsy field to reveal novel insights into seizures and epilepsy. Moreover, we discuss how caution is needed in regards to rodent strain or even animal vendor choice, and how this can significantly influence seizure and epilepsy parameters in unexpected ways. This is particularly critical in decisions regarding the strain of choice used in generating mice with targeted deletions of genes. Finally, we discuss the role of environment (at vendor and/or laboratory) and epigenetic factors for inter- and intrastrain differences and how such differences can affect the expression of seizures and the animals' performance in behavioral tests that often accompany acute and chronic seizure testing.

Characterization of hexose transporters in Yarrowia lipolytica reveals new groups of Sugar Porters involved in yeast growth

Sugar assimilation has been intensively studied in the model yeast S. cerevisiae, and for two decades, it has been clear that the homologous HXT genes, which encode a set of hexose transporters, play a central role in this process. However, in the yeast Yarrowia lipolytica, which is well-known for its biotechnological applications, sugar assimilation is only poorly understood, even though this yeast exhibits peculiar intra-strain differences in fructose uptake: some strains (e.g., W29) are known to be slow-growing in fructose while others (e.g., H222) grow rapidly under the same conditions. Here, we retrieved 24 proteins of the Sugar Porter family from these two strains, and determined that at least six of these proteins can function as hexose transporters in the heterologous host Saccharomyces cerevisiae EBY.VW4000. Transcriptional studies and deletion analysis in Y. lipolytica indicated that two genes, YHT1 and YHT4, are probably the main players in both strains, with a similar role in the uptake of glucose, fructose, and mannose at various concentrations. The other four genes appear to constitute a set of ‘reservoir’ hexose transporters with an as-yet unclear physiological role. Furthermore, through examining Sugar Porters of the entire Yarrowia clade, we show that they constitute a dynamic family, within which hexose transport genes have been duplicated and lost several times. Our phylogenetic analyses support the existence of at least three distinct evolutionary groups of transporters which allow yeasts to grow on hexoses. In addition to the well-known and widespread Hxt-type transporters (which are not essential in Y. lipolytica), we highlight a second group of transporters, represented by Yht1, which are phylogenetically related to sensors that play a regulatory role in S. cerevisiae, and a third group, represented by Yht4, previously thought to contain only high-affinity glucose transporters related to Hgt1of Kluyveromyces lactis.

The pilocarpine model of temporal lobe epilepsy: Marked intrastrain differences in female Sprague-Dawley rats and the effect of estrous cycle.

Rat strains such as Sprague-Dawley (SD) or Wistar are widely used in epilepsy research, including popular models of temporal lobe epilepsy in which spontaneous recurrent seizures (SRS), hippocampal damage, and behavioral alterations develop after status epilepticus (SE). Such rats are randomly outbred, and outbred strains are known to be genetically heterogeneous populations with a high intrastrain variation. Intrastrain differences may be an important reason for discrepancies between studies from different laboratories, but the extent to which such differences affect the development of seizures, neurodegeneration, and psychopathology in post-SE models of epilepsy has received relatively little attention. In the present study, we induced SE by systemic administration of pilocarpine (following pretreatment with lithium) in SD rats from different breeders (Harlan, Charles River [CRL], Taconic) as well as different breeding locations of the same breeder (Harlan-Winkelmann [HW] in Germany vs. Harlan Laboratories [HL] in the Netherlands). Some experiments were also performed in Wistar rats. Pilocarpine was administered by a ramp-up dosing protocol that allows determining interindividual differences in susceptibility to the convulsant. Marked intrastrain differences in induction of SE and its long-term consequences were found. Sprague-Dawley rats from HW were significantly more sensitive to SE induction than all other SD substrains. The majority of SD rats from different vendors developed SRS after SE except SD rats from HL. The CRL-SD rats markedly differed in basal behavior and SE-induced behavioral alterations from other SD substrains. Susceptibility to pilocarpine was hardly affected by the estrous cycle. The marked intrastrain differences provide an interesting tool to study the impact of genetic and environmental factors on seizure susceptibility, epileptogenesis, and relationship between behavior and epilepsy and vice versa.

English

The objective of this study was to examine the effect of interacting conditions of water stress (0.995-0.96 water activity; aw), elevated temperature (25-37°C) and CO2 (350, 1000 ppm) on growth and sporulation of strains of three entomogenous fungi, Beauveria bassiana, Metarhizium anisopliae and Isaria farinosa. Subsequently, using bioassay systems with locust (Schistocerca gregaria), we examined the effect of elevated CO2 (control, 350; 650; 1000 ppm CO2) on efficacy of strains of all three species and used crickets (Acheta domesticus) to examine interacting conditions of elevated temperature and CO2 at two relative humidities (25-35°C; 350, 1000 ppm CO2; 96, 98 and >99% RH) on efficacy of a strain of B. bassiana for the first time. The 3-way interacting factors had a significant effect on growth of the strains of all three species, especially at 35-37°C and 0.96-0.98 aw and 1000 ppm CO2. Under these conditions, only one strain of B. bassiana and M. anisopliae was able to grow at a reduced rate as compared to the controls. No strain of I. farinosa was able to grow at 35-37°C either in normal air or in elevated CO2 at 0.995-0.96 aw showing a high level of sensitivity to these interacting factors. Sporulation of the three strains of each species was also significantly affected by these three-way environmental interactions. There were some intra-strain differences and in most cases for the three species, water stress (0.98-0.96 aw) at 35-37°C and 1000 ppm CO2 resulting in either no sporulation or no growth. One strain of M. anisopliae (Ma 29) was particularly tolerant at 0.96 aw at 37°C and 1000 ppm CO2. Bioassays with the S. gregaria showed when CO2 was elevated from 350 to 650 and to 1000 ppm, the relative virulence of two strains of each species was reduced over a 6-day temporal study. Further studies with B. bassiana in a detailed bioassay using crickets under three way abiotic interactions (25-35°C, 99-96% RH and 350 or 1000 ppm CO2) showed that virulence was decreased with no efficacy occurring at 30-35°C and 1000 ppm CO2 at 96% RH. This study suggests that climate change factors could have a profound impact on the efficacy of such biocontrol agents and thus have major implications for pest control using such approaches. Key words: Water stress, temperature, elevated CO2, growth, sporulation, entomopathogenic fungi, pest control.

Comparative study of water and temperature relations of growth and T-2/HT-2 toxin production by strains of Fusarium sporotrichioides and Fusarium langsethiae

This study has compared the effect of water activity (aw, 0.995-0.93) and two temperatures (15, 25 °C) on the growth and HT-2 and T-2 toxin production by three strains of Fusarium sporotrichioides and Fusarium langsethiae in an oat-based medium. There were very little intra-strain differences for each of the species in relation to growth at 0.995-0.93 aw and 15 and 25 °C. Inter-species comparisons showed that at 0.995 and 0.98 aw growth was similar at both temperatures. However, under water stress at 0.95 and 0.93 aw F. sporotrichioides strains were more tolerant than those of F. langsethiae. Statistical analyses showed that aw and temperature were significant factors for both species, while strain differences were not (P=0.05). In contrast, the patterns of production of T-2 and HT-2 toxins were very different from that for growth. For F. sporotrichioides, a generally higher amount of toxin was produced at 15 than at 25 °C, with a maximum produced at 0.995 aw, while the production decreased with water stress. Interestingly, maximum production of T-2+HT-2 toxin was achieved by F. langsethiae at 0.98 aw and 25 °C, with very little produced at 0.93 aw. Statistically, aw was the most significant (P=0.05) factor for the mean of the three strains of both species with a significant inter-species difference in terms of toxin production. This was confirmed by the changes in the ratio of HT-2:T-2 toxin in relation to the aw × temperature conditions studied. These results are discussed in the context of the effect of environment on the relative importance of these two species in contaminating oats with these toxins.

Marked strain and substrain differences in induction of status epilepticus and subsequent development of neurodegeneration, epilepsy, and behavioral alterations in rats: Strain and substrain differences in an epilepsy model in rats

Outbred rat strains such as Sprague-Dawley (SD) or Wistar are widely used in epilepsy models, including popular models of temporal lobe epilepsy in which spontaneous recurrent seizures (SRS), hippocampal damage, and behavioral alterations develop after status epilepticus (SE). Such rats are randomly outbred, hence allelic variations can occur across separate colonies, so that outbred rats from different sources may have little in common with each other besides their names and similarities in pelage. Although such intrastrain differences may be an important reason for discrepancies between studies from different laboratories, the extent to which such differences affect development of seizures, neurodegeneration, and psychopathology in post-SE models of epilepsy has received little attention as yet. In the present study, we induced SE by sustained electrical stimulation of the basolateral amygdala in SD and Wistar rats from different breeders (Harlan, Charles River, Janvier, Taconic) as well as different breeding locations of the same breeder (Harlan-Winkelmann in Germany vs. Harlan in the Netherlands). Several marked inter- and intrastrain differences in induction of SE and its long-term consequences were found. Wistar rats from different vendors were all strikingly less sensitive to SE induction than SD rats from Harlan-Winkelmann or Harlan. Within the SD strain, SD rats from Charles River exhibited markedly lower sensitivity to SE induction than all other groups of SD rats. The majority of SD rats from different vendors developed SRS after SE except SD rats from Charles River. The latter rats also markedly differed in basal behavior, SE-induced behavioral alterations and neurodegeneration from other SD substrains. These marked inter- and intrastrain differences provide an interesting tool to study the impact of genetic and environmental factors on seizure susceptibility, epileptogenesis, and the relationship between behavior and epilepsy and vice versa.

Central effects of a local inflammation in three commonly used mouse strains with a different anxious phenotype

As in humans, genetic background in rodents may influence a peculiar set of behavioural traits such as sensitivity to pain and stressors or anxiety-related behaviours. Therefore, we tested the hypothesis that mice with different genetic backgrounds [outbred (CD1), inbred (C57BL/6J) and hybrid (B6C3F1) adult male mice] display altered reactivity to pain, stress and anxiety related behaviours.We demonstrated that B6C3F1 mice displayed the more anxious phenotype with respect to C57BL/6J or CD1 animals, with the latter being the less anxious strain when tested in an open field and on an elevated plus maze. No difference was observed across strains in thermal sensitivity to a radiant heat source. Mice were then treated with a sub-plantar injection of the inflammatory agent Complete Freund's Adjuvant (CFA), 24h later they were hyperalgesic with respect to saline exposed animals, irrespective of strain. We then measured intra-strain differences and CFA-induced inter-strain effects on the expression of various genes with a recognized role in pain and anxiety: BDNF, IL-6, IL-1β, IL-18 and NMDA receptor subunits in the mouse thalamus, hippocampus and hypothalamus. The more anxious phenotype observed in B6C3F1 hybrid mice displayed lower levels of BDNF mRNA in the hippocampus and hypothalamus when compared to outbred CD1 and C57BL/6J inbred mice. CFA led to a general decrease in central gene expression of the evaluated targets especially in CD1 mice, while BDNF hypothalamic downregulation stands out as a common effect of CFA in all three strains evaluated.

Temperature and water activity effects on production of T-2 and HT-2 by Fusarium langsethiae strains from north European countries

This study has examined the effect of ecophysiological factors, water activity (a w , 0.995–0.90) and temperature (10–37 °C), on the T-2 and HT-2 toxins production by Fusarium langsethiae. Two dimensional profiles for optimum and marginal conditions have been built for two strains from each of four northern European countries (UK, Norway, Sweden, Finland) on an oat-based medium. This showed that the optimum a w and temperature conditions for T-2 + HT-2 production was between 0.98–0.995, and 20–30 °C respectively. Kruskal–Wallis analysis of ranks showed a statistically significant differences between the different a w levels examined ( P < 0.001) but no significant effect of the temperatures examined. The ratio of HT-2/T-2 was investigated and non-uniform distribution of HT-2 toxin was found under different ecological conditions. No statistically significant differences were found for the mean toxin production between strains from the different countries. Intra-strain differences in toxin production was only found for those from Finland ( P-value = 0.0247). The growth/no growth and toxin/no toxin conditions in relation to a w x temperature have been constructed for the first time. This knowledge will be useful in developing prevention strategies to minimise T-2 and HT-2 toxin contamination by strains of F. langsethiae on important small grain cereals.

Intrastrain differences in seizure susceptibility, pharmacological response and basal neurochemistry of Wistar rats

Reliable well-characterised animal models of seizures are necessary in order to better understand the underlying pathophysiological mechanisms as well as to screen potential anticonvulsant drugs. We currently use the focal pilocarpine model as an acute limbic seizure model. Due to breeding problems at the vendor, and apparent changes in pilocarpine-induced seizure susceptibility, we were forced to change breeding locations and vendors over a period of 2 years. Male Wistar rats were either purchased from two breeding locations of Charles River Laboratories (France and Germany), or obtained from Harlan Laboratories (The Netherlands). In the present retrospective study we evaluated the impact of these vendor changes on ketamine dosing to establish anaesthesia, on pilocarpine-induced seizure susceptibility, and on basal extracellular hippocampal noradrenaline, dopamine, serotonin, gamma-amino butyric acid, and glutamate levels of all pilocarpine-treated rats included in our studies. Significant differences were present in all of the parameters analyzed. This study clearly illustrates that intrastrain differences do exist from one vendor/breeding location to another, or even between rats from the same breeding location.

Differential stress-induced alterations of colonic corticotropin-releasing factor receptors in the Wistar Kyoto rat

BACKGROUND A growing body of data implicates increased life stresses with the initiation, persistence and severity of symptoms associated with functional gut disorders such as irritable bowel syndrome (IBS). Activation of central and peripheral corticotropin-releasing factor (CRF) receptors is key to stress-induced changes in gastrointestinal (GI) function. METHODS This study utilised immunofluorescent and Western blotting techniques to investigate colonic expression of CRF receptors in stress-sensitive Wistar Kyoto (WKY) and control Sprague Dawley (SD) rats. KEY RESULTS No intra-strain differences were observed in the numbers of colonic CRFR1 and CRFR2 positive cells. Protein expression of functional CRFR1 was found to be comparable in control proximal and distal colon samples. Sham levels of CRFR1 were also similar in the proximal colon but significantly higher in WKY distal colons (SD: 0.38 +/- 0.14, WKY: 2.06 +/- 0.52, P < 0.01). Control levels of functional CRFR2 were similar between strains but sham WKYs samples had increased CRFR2 in both the proximal (SD: 0.88 +/- 0.21, WKY: 1.8 +/- 0.18, P < 0.001) and distal (SD: 0.18 +/- 0.08, WKY: 0.94 +/- 0.32, P < 0.05) regions. Exposure to open field (OF) and colorectal distension (CRD) stressors induced decreased protein expression of CRFR1 in SD proximal colons, an effect that was blunted in WKYs. CRD stimulated decreased expression of CRFR2 in WKY rats alone. Distally, CRFR1 is decreased in WKY rats following CRD but not OF stress without any apparent changes in SD rats. CONCLUSIONS & INFERENCES This study demonstrates that psychological and physical stressors alter colonic CRF receptor expression and further support a role for local colonic CRF signalling in stress-induced changes in GI function.

Differential SPECT Activation Patterns Associated with PASAT Performance May Indicate Frontocerebellar Functional Dissociation in Chronic Mild Traumatic Brain Injury

Patients with mild traumatic brain injury (TBI) often complain of cognitive fatigue during the chronic recovery phase. The Paced Auditory Serial Addition Test (PASAT) is a complex psychologic measure that may demonstrate subtle deficiencies in higher cognitive functions. The purpose of this study was to investigate the brain activation of regional cerebral blood flow (rCBF) with PASAT in patients with mild TBI to explore mechanisms for the cognitive fatigue. Two groups consisting of 15 patients with mild TBI and 15 healthy control subjects underwent (99m)Tc-ethylene cysteine dimer SPECT at rest and during PASAT on a separate day. Cortical rCBF was extracted using a 3-dimensional stereotactic surface projection and statistically analyzed to identify areas of activation, which were compared with PASAT performance scores. Image analysis demonstrated a difference in the pattern of activation between patients with mild TBI and healthy control subjects. Healthy control subjects activated the superior temporal cortex (Brodmann area [BA] 22) bilaterally, the precentral gyrus (BA 9) on the left, and the precentral gyrus (BA 6) and cerebellum bilaterally. Patients with mild TBI demonstrated a larger area of supratentorial activation (BAs 9, 10, 13, and 46) but a smaller area of activation in the cerebellum, indicating frontocerebellar dissociation. Patients with mild TBI and cognitive fatigue demonstrated a different pattern of activation during PASAT. Frontocerebellar dissociation may explain cognitive impairment and cognitive fatigue in the chronic recovery phase of mild traumatic brain injury.

GROWTH AND PHOTOPROTECTION IN THREE DINOFLAGELLATES (INCLUDING TWO STRAINS OF ALEXANDRIUM TAMARENSE) AND ONE DIATOM EXPOSED TO FOUR WEEKS OF NATURAL AND ENHANCED ULTRAVIOLET-B RADIATION(1).

Long-term growth response to natural solar radiation with enhanced ultraviolet-B (UVB) exposure was examined in two species of dinoflagellates [Alexandrium tamarense (M. Lebour) Balech, At, and Heterocapsa triquetra (Ehrenb.) F. Stein, Ht], including two strains of A. tamarense, one from Spain and another from UK, and one diatom species (Thalassiosira pseudonana Hasle et Heimdal). We examined whether variable photoprotection (mycosporine-like amino acids [MAAs] and xanthophyll-cycle pigments) affected photosynthetic performance, phytoplankton light absorption, and growth. Growth rate was significantly reduced under enhanced UVB for the UK strain of At and for Ht (both grew very little) as well as for the diatom (that maintained high growth rates), but there was no effect for the Spanish strain of At. MAA concentration was high in the dinoflagellates, but undetectable in the diatom, which instead used the xanthophyll cycle for photoprotection. The highest cell concentrations of MAAs and photoprotective pigments were observed in the UK strain of At, along with lowest growth rates and Fv /Fm , indicating high stress levels. In contrast, the Spanish strain showed progressive acclimation to the experimental conditions, with no significant difference in growth between treatments. Increase in total MAAs followed linearly the cumulative UVB of the preceding day, and both total and primary MAAs were maintained at higher constitutive levels in this strain. Acclimation to enhanced UVB in the diatom resulted in an increase in PSII activity and reduction in nonphotochemical quenching, indicating an increased resistance to photoinhibition after a few weeks. All four species showed increased phytoplankton light absorption under enhanced UVB. Large intrastrain differences suggest a need to consider more closely intraspecific variability in UV studies.

IMPORTANCE OF CYP2D3 IN POLYMORPHISM OF DIAZEPAMP-HYDROXYLATION IN RATS

Diazepam was metabolized to three primary metabolites, 3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam. Our previous studies reported metabolic position-specific inter- or intrastrain differences in diazepam metabolism among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. Especially, there were marked ( approximately 300 fold) inter- or intrastrain differences in diazepam p-hydroxylation activity at low concentration of substrate. In this study, we investigated the enzyme that catalyzes diazepam p-hydroxylation. The activity toward diazepam p-hydroxylation was inhibited by anti-cytochrome P450 2D (CYP2D) antibody, suggesting that this activity was catalyzed by CYP2D isoforms. Comparing the expression levels of the CYP2D subfamily in liver microsomes from various strains of rats using anti-CYP2D2 antibody, we found that there was a band of protein that was consistent with the phenotype of diazepam p-hydroxylation. N-terminal amino acid sequences of the specific protein exactly corresponded to those of CYP2D3, indicating that CYP2D3 might be involved in diazepam p-hydroxylation. Moreover, using rat CYP2D isoforms expressed in yeast, we tested CYP2Ds to catalyze diazepam p-hydroxylation. CYP2D1 and CYP2D2 practically did not participate in diazepam metabolism. On the other hand, diazepam p-hydroxylation was catalyzed by CYP2D3. CYP2D4 had high activity toward diazepam N-desmethylation, but not p-hydroxylation. In conclusion, the polymorphic expression of CYP2D3 caused the inter- or intrastrain differences in diazepam p-hydroxylation among rat strains or individuals.

Intra-strain dioxin sensitivity and morphometric effects in swim-up rainbow trout ( Oncorhynchus mykiss)

Inter and intra-specific differences in sensitivity of early life stage salmonids to 2,3,7,8-TCDD exposure have been reported, but intra-strain differences have not been found in the literature. Our results indicate that intra-strain variability in terms of embryo mortality (LD50) is small in Eagle Lake strain of rainbow trout, LD50 values ranging from 285 to 457 pg TCDD egg g −1. These results confirm Eagle Lake as a less sensitive strain within rainbow trout, and do not indicate overlap with reported LD50 values for brook or lake trout. Our results also demonstrate that although generalized edema in regions including the yolk-sac are frequently associated with mortality following dioxin exposure, not all edematous fish die. We detected dose-dependent decreases in cranial length, eye diameter, mass, and total length ( P<0.05) in viable swim-up rainbow trout. These effects are presumed to indicate more subtle dose-dependent disruptions of the viteline vein vasculature and, therefore, in access to energy sources. A tendency for dose-dependent decrease in liver glycogen reserves concurred with previous results on salmonids and with the well described TCDD-induced alterations in intermediate metabolism of rats and chicken embryos (wasting syndrome). This syndrome could be contributing to the reduced growth that we observed.

Strain and colony differences in the neurotoxic sequelae of MK-801 visualized with the amino-cupric-silver method

The strain and sex of a species under investigation may influence the animal's physiological response to a variety of stimuli. Strain and sex differences are important considerations when evaluating animal models. In the rodent MK-801 model of schizophrenia, degenerative changes occur widely in the main olfactory system and in a number of cortical brain regions. In the present report, we compare the effects of MK-801 neurotoxicity in two strains of female rats and also two lines within each strain. The magnitude and regional extent of the neurodegeneration detected with the amino-cupric-silver method varied markedly both between the Sprague-Dawley and Wistar rat strains and also between two lines derived from each strain. For example, terminal degeneration occurred in layer VI of somatosensory cortex and the central extended amygdala in Sprague-Dawley but not Wistar rats. Moreover, MK-801 treatment led to somatodendritic degeneration in the dentate gyrus of the dorsal hippocampus and basolateral amygdala in Wistar rats from Charles River Laboratories but not those from Ferreyra Institute. There are thus both strain and intrastrain differences in the magnitude of the neurodegenerative response to MK-801 treatment. The differing neurotoxicity of MK-801 between rat strains and between lines within a strain may reflect genetic variation and/or differences in hepatic biotransformation and thus the bioavailability of the drug between strains and lines within a strain.

Mucin Apoprotein Expression in COPD

Mucins, which are complex glycoproteins that provide the viscoelastic properties of mucus that are essential for the protection of the airways, are characterized by a variable-number tandem repeats (VNTR) region that may undergo alternate splicing during transcription. Such transcripts may yield multiple proteins via diverse post-translational modifications involving glycosylation (within each VNTR). Fifteen distinct mucin genes have been identified, with several mapping to chromosomal clusters (ie, 7q22 and 11p15.5), possibly having evolved by gene duplication. The deduced protein sequences can be subdivided into both membrane-associated mucins and secreted mucins. Membrane-associated mucins consist of cytoplasmic, transmembrane, and extracellular domains. The membrane-associated mucins MUC1, MUC4, and MUC11 have been localized to the lung. In addition to VNTRs, secreted mucins possess repeated cysteine-rich D-domains (which are important in polymerization). Secreted mucins that are localized to the lung include MUC2 (in cells with and without secretory granules), MUC5AC (in surface and submucosal mucous cells), MUC5B and MUC8 (in submucosal mucous cells), and MUC7 (in submucosal serous cells). Currently, little is known about the regulation of mucins in COPD patients. Recent studies with acrolein and cigarette smoke have suggested that MUC5AC is inducible (accompanied by epidermal growth factor [EGF] ligand formation and the activation of EGF receptor-dependent pathways), whereas MUC5B is constitutively expressed (increasing through gland enlargement). Similarly, little is known about the genetic determinants that control mucus hypersecretion, but preliminary findings in animal models suggest that intrastrain differences in acrolein-induced mucin formation are amenable to genetic analysis. As our understanding of the functional genomics of mucin biology increases, further clinical targets and therapeutic strategies are likely to emerge.