ADAMTS5 deficiency in mice does not affect cardiac function

The aggrecanase ADAMTS5 (A Disintegrin and Metalloproteinase with ThromboSpondin type 1 motifs, member 5) and the cleavage of its substrate versican have been implicated in the development of heart valves. Furthermore, ADAMTS5 deficiency was shown to protect against diet‐induced obesity, a known risk factor for cardiovascular disease. Therefore, in this study, we investigated the potential role of ADAMTS5 in cardiac function using ADAMTS5‐deficient (Adamts5^?/?) mice and their wild‐type (Adamts5^+/+) counterparts exposed to a standard‐fat or a high‐fat diet (HFD). Eight‐weeks‐old Adamts5^?/? and Adamts5^+/+ mice were exposed to each diet for 15 weeks. Cardiac function and electrophysiology were analyzed by transthoracic echocardiogram and electrocardiogram at the end of the study. Cleavage of versican, as detected by the appearance of the DPEEAE neo‐epitope on western blotting with protein extracts, was defective in the heart of HFD‐treated Adamts5^?/? as compared with Adamts5^+/+ mice. ADAMTS5 deficiency led to statistically significant increases in diastolic posterior wall thickness (0.94 ± 0.023 vs. 0.82 ± 0.036 mm; P = 0.0056) and left ventricle volume (47 ± 4.5 vs. 31 ± 2.5 μL; P = 0.0043) in comparison to Adamts5^+/+ mice, but only in animals on a HFD. Cardiac function parameters such as ejection fraction, fractional shortening, and stroke volume were unaffected by ADAMTS5 deficiency or diet. Electrocardiogram analysis revealed no ADAMTS5‐specific changes in either diet group. Thus, in the absence of ADAMTS5, cleavage of versican in the cardiac extracellular matrix is impaired, but cardiac function, even upon exposure to a HFD, is not markedly affected.     

Innate immunity: squelching anti-viral signalling with NLRX1

Innate immunity to viruses involves receptors such as RIG-I, which senses viral RNA and triggers a signalling pathway involving the outer mitochondrial membrane protein MAVS. Recent work has identified that NLRX1, a member of another class of innate immune receptors, sequesters MAVS away from RIG-I and thereby prevents mitochondrial anti-viral immunity.     

Axl deficiency does not affect adipogenesis or adipose tissue development

To evaluate a potential role of Axl, the high-affinity receptor of growth arrest-specific protein 6 (GAS6) in adiposity, murine embryonic fibroblasts (MEF) derived from mice with genetic deficiency of Axl (Axl(-/-)) or wild-type littermates (Axl(+/+)) were differentiated into mature adipocytes. In addition, Axl(-/-) and Axl(+/+) mice were kept on standard fat diet (SFD) or on high-fat diet (HFD) for 15 weeks. Deficiency of Axl in MEF did not affect differentiation, as shown by a similar uptake of Oil Red O and expression of the adipogenic markers aP2 and peroxisome proliferator activator receptor γ (PPARγ) at the end of the differentiation. In the first 7 weeks of HFD feeding, Axl(-/-) mice gained less weight than their wild-type littermates. Weight gain for both genotypes on either SFD of HFD over 15 weeks was, however, not significantly different, resulting in comparable body weights, as well as subcutaneous (s.c.) and gonadal (GON) fat mass. Adipocyte size in the fat tissues was not affected by Axl deficiency. Gene expression analysis indicated that the absence of Axl in vivo may be compensated for by the other TAM family members Mer and Tyro3. Glucose and insulin tolerance tests (ITT) in Axl(-/-) and Axl(+/+) mice did not reveal significant differences in glucose homeostasis. Thus, Axl deficiency had no significant effect on adipogenesis in vitro or in vivo.     

Nutritional copper deficiency does not affect sponge granuloma formation in the rat

Sponge granuloma formation was compared in copper-deficient and copper-sufficient rats following feeding of respective diets for 20, 40, or 60 d. Body weight, total blood hemoglobin, and activities of ceruloplasmin and Cu, Zn-superoxide dismutase in plasma were monitored to ascertain copper deficiency. Mean granuloma weights (mg +/- SEM) in copper-deficient and copper-sufficient groups of rats, respectively, were as follows: 37 +/- 2 and 38 +/- 2 after 20 d, 22 +/- 2 and 23 +/- 2 after 40 d, and 19 +/- 1 and 21 +/- 1 after 60 d on respective diets. Thus, nutritional copper deficiency did not have an effect on sponge granuloma formation in the rat.     

Genetic vitamin E deficiency does not affect MPTP susceptibility in the mouse brain

Oxidative stress is involved in the degeneration of the nigrostriatal dopaminergic system in Parkinson's disease (PD). Vitamin E (alpha-tocopherol) is a potent antioxidant in the cell membrane that can trap free radicals and prohibit lipid peroxidation. The retention and secretion of vitamin E are regulated by alpha-tocopherol transfer protein (TTP) in the brain and liver. Dysfunction of TTP results in systemic deficiency of vitamin E in humans and mice, and increased oxidative stress in mouse brain. In this study, we investigated the effect of vitamin E deficiency in PD development by generating an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD using TTP knockout (TTP-/-) mice. Vitamin E concentration in the brains of TTP+/- mice was half that in TTP+/+ mice, and in TTP-/- mice, was undetectable. MPTP treatment tended to decrease striatal dopamine, but the effect was comparable and not significant in any of the three genotypes. Furthermore, the extent of loss of dopaminergic cell bodies in the substantia nigra did not differ among the groups. One the other hand, oral administration of vitamin E resulted in the partial protection of striatal dopaminergic terminals against MPTP toxicity. Our results suggest that vitamin E does not play a major protective role in MPTP-induced nigrostriatal dopaminergic neurodegeneration in the brain.     

Experimental evolution reveals that population density does not affect moth signalling behaviour and antennal morphology

Population density can play a vital role in determining investment in reproductive behaviours and morphologies of invertebrates. Males reared in high-density environments, where competition is high but difficulties in locating mates are low, may invest more in reproductive structures associated with sperm competition such as testes, at the expense of those traits associated with mate location, such as antennae. In species where females advertise for mates, such as most moths, a high-density environment may also lead to a reduction in pheromonal signalling (calling) length and frequency as a result of high mate abundance. While such responses have been shown at the phenotypically plastic level in moths, heritable evolutionary adaptations have seldom been tested, and studies of how population density influences pheromone signalling strategies are scarce. Here we use behavioural assays and scanning electron microscopic measurements to test whether larval population density influences, at the genetic level, the ability of males to locate females and male investment into antennal morphology, in addition to its effect on the frequency and duration of female calling. We used two replicated populations of the Indian meal moth Plodia interpunctella that had experimentally evolved under high or low population densities for 35 generations. We found no significant divergence in antennal morphology or mate acquisition behaviours between the two density populations. These findings suggest that although population density has the ability to create plastic changes in both morphological and behavioural traits, this factor alone is unlikely to be causing evolutionary change in male and female signalling in this species.     

SOCS-1 deficiency does not prevent diet-induced insulin resistance

Obesity is associated with inflammation and increased expression of suppressor of cytokine signaling (SOCS) proteins, which inhibit cytokine and insulin signaling. Thus, reducing SOCS expression could prevent the development of obesity-induced insulin resistance. Using SOCS-1 knockout mice, we investigated the contribution of SOCS-1 in the development of insulin resistance induced by a high-fat diet (HFD). SOCS-1 knockout mice on HFD gained 70% more weight, displayed a 2.3-fold increase in epididymal fat pads mass and increased hepatic lipid content. This was accompanied by increased mRNA expression of leptin and the macrophage marker CD68 in white adipose tissue and of SREBP1c and FAS in liver. HFD also induced hyperglycemia in SOCS-1 deficient mice with impairment of glucose and insulin tolerance tests. Thus, despite the role of SOCS proteins in obesity-related insulin resistance, SOCS-1 deficiency alone is not able to prevent insulin resistance induced by a diet rich in fat.     

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer’s disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT^−/− mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer’s-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.     

Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses

If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak.     

Cbp deficiency alters Csk localization in lipid rafts but does not affect T-cell development

The ubiquitously expressed transmembrane adaptor Csk-binding protein (Cbp) recruits Csk to lipid rafts, where the latter exerts its negative regulatory effect on the Src family of protein tyrosine kinases. We have inactivated Cbp in the mouse germ line. In contrast to Csk gene inactivation, which leads to embryonic lethality and impaired T-cell development, Cbp-deficient mice were viable and exhibited normal T-cell development but with an increased thymocyte population. In the absence of Cbp, the amount of Csk that localizes to the lipid rafts was greatly reduced. Interestingly, this altered lipid raft localization of Csk did not lead to any detectable biochemical or functional defect in T cells. The T-cell receptor-induced intracellular calcium flux, cell proliferation, and cytokine secretion were not affected by the absence of Cbp. Peripheral T-cell tolerance to superantigen SEB was also largely intact in Cbp-deficient mice. Thus, Cbp is dispensable for T-cell development and activation.     

Targeted disruption of the testicular SPAG5/deepest protein does not affect spermatogenesis or fertility

In an effort to define the molecular basis for morphogenesis of major sperm tail structures, including outer dense fibers, we recently cloned the Spag5 gene by virtue of its strong and specific leucine-zipper-mediated interaction with Odf1, the 27-kDa major outer dense fiber protein. Spag5 is expressed during meiosis and in round spermatids and is similar, if not identical, to Deepest, a putative spindle pole protein. Here we report the disruption of the Spag5 gene by homologous recombination. Spag5-null mice lack Spag5 mRNA and protein. However, male mice are viable and fertile. Analysis of the process of spermatogenesis and sperm produced in Spag5-null mice did not reveal a major phenotype as a consequence of the knockout event. This result suggests that if Spag5 plays a role in spermatogenesis it is likely compensated for by unknown proteins.     

DNA demethylation induced by 5-azacytidine does not affect fragile X expression.

Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treated cells. Under conditions where 5-azacytidine was found to inhibit fragile X expression, no DNA demethylation was observed. At the time when demethylation did occur, fragile X expression was not affected. These results strongly indicate that DNA demethylation is not involved in fragile X expression.     

The absence of nidogen 1 does not affect murine basement membrane formation

Nidogen 1 is a highly conserved protein in mammals, Drosophila melanogaster, Caenorhabditis elegans, and ascidians and is found in all basement membranes. It has been proposed that nidogen 1 connects the laminin and collagen IV networks, so stabilizing the basement membrane, and integrates other proteins, including perlecan, into the basement membrane. To define the role of nidogen 1 in basement membranes in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen 1 mRNA nor protein. Surprisingly, they show no overt abnormalities and are fertile, their basement membrane structures appearing normal. Nidogen 2 staining is increased in certain basement membranes, where it is normally only found in scant amounts. This occurs by either redistribution from other extracellular matrices or unmasking of nidogen 2 epitopes, as its production does not appear to be upregulated. The results show that nidogen 1 is not required for basement membrane formation or maintenance.     

NLRC5 deficiency does not influence cytokine induction by virus and bacteria infections

Nucleotide-binding domain and leucine rich repeat containing gene family receptors (NLRs) are cytosolic proteins that respond to a variety of pathogen and host components to induce inflammatory cytokines. NLRC5 is a recently identified member of the NLR family that has been implicated in positive and negative regulation of antiviral innate immune responses. To clarify whether NLRC5 controls antiviral innate immunity in vivo, we generated NLRC5-deficient mice. Macrophages and dendritic cells derived from NLRC5-deficient mice induced relatively normal levels of IFN-β, IL-6, and TNF-α after treatment with RNA viruses, DNA viruses, and bacteria. The serum cytokine levels after polyinosinic-polycytidylic acid infection were also comparable between control and NLRC5-deficient mice. NLRC5 overexpression promoted IL-1β production via caspase-1, suggesting that NLRC5 constitutes an inflammasome. However, there was no reduction of IL-1β in NLRC5-deficient cells in response to known inflammasome activators, suggesting that NLRC5 controls IL-1β production through an unidentified pathway. These findings indicate that NLRC5 is dispensable for cytokine induction in virus and bacterial infections under physiologic conditions.     

Hibernation does not affect memory retention in bats

Long-term memory can be critically important for animals in a variety of contexts, and yet the extreme reduction in body temperature in hibernating animals alters neurochemistry and may therefore impair brain function. Behavioural studies on memory impairment associated with hibernation have been almost exclusively conducted on ground squirrels (Rodentia) and provide conflicting results, including clear evidence for memory loss. Here, we for the first time tested memory retention after hibernation for a vertebrate outside rodents—bats (Chiroptera). In the light of the high mobility, ecology and long life of bats, we hypothesized that maintenance of consolidated memory through hibernation is under strong natural selection. We trained bats to find food in one out of three maze arms. After training, the pre-hibernation performance of all individuals was at 100 per cent correct decisions. After this pre-test, one group of bats was kept, with two interruptions, at 7°C for two months, while the other group was kept under conditions that prevented them from going into hibernation. The hibernated bats performed at the same high level as before hibernation and as the non-hibernated controls. Our data suggest that bats benefit from an as yet unknown neuroprotective mechanism to prevent memory loss in the cold brain.     

Prostacyclin does not affect insulin secretion in humans

The effects of Prostacyclin (PGI₂) infusion on insulin secretion and glucose tolerance were investigated in 7 healthy subjects. PGI₂ infusion caused no statistically significant changes of either glucose or insulin concentration, over the range 2.5–20 ng/Kg/min. A constant PGI₂ infusion (10 ng/Kg/min) did not inhibit acute insulin responses to a glucose (20 g i.v.) pulse (response before PGI₂ = 612±307%; during PGI₂ = 515±468%, mean ± SD, mean change 3–5 min insulin, % basal; P=NS). Glucose disappearance rates were similar after the first and second glucose pulse.Thus, in contrast to PGE₂, PGI₂ does not affect insulin secretion nor glucose disposal at doses producing platelet and vascular changes. It is hypothesized that an altered PGI₂/PGE₂ balance in diabetes may represent a link between vascular, platelet and metabolic changes.     

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1^−/− mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1^+/+ and Npc1^−/− microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1^−/− microglia is ~ 60% lower than in Npc1^+/+ microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1^−/− and Npc1^+/+ neurons in microglia-neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1^−/− microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia-neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease.     

Ghrelin deficiency does not influence feeding performance

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor that is synthesized predominantly in the stomach. Previous studies demonstrated that ghrelin stimulates growth hormone release and food intake. These data suggested that antagonism of ghrelin could serve as a useful treatment for eating disorders and obesity. To study the role of endogenous ghrelin in feeding performance further, we generated ghrelin-deficient (ghrl(-/-)) mice. Unexpectedly, ghrl(-/-) mice exhibited normal growth, cumulative food intake, reproduction, histological characters, and serum parameters. There were no differences in feeding patterns between ghrl(+/+) and ghrl(-/-) mice. Ghrl(-/-) mice displayed normal responses to scheduled feedings as seen for ghrl(+/+) mice. Memory-related feeding performances of ghrl(-/-) mice were indistinguishable from ghrl(+/+) littermates. These data indicate that ghrelin is not critical for feeding performance.