Functional analysis of novel mutations in y(+)LAT-1 amino acid transporter gene causing lysinuric protein intolerance (LPI)

Lysinuric protein intolerance (LPI; MIM 222700) is an autosomal recessive disorder characterized by defective transport of the cationic amino acids lysine, arginine and ornithine at the basolateral membrane of the polar epithelial cells in the intestine and renal tubules, and by hyperammonemia after high-protein meals. LPI is caused by mutations in the SLC7A7 (solute carrier family 7, member 7) gene encoding y(+)LAT-1 (y(+)L amino acid transporter-1), which co-induces together with 4F2 heavy chain (4F2hc) system y(+)L in Xenopus oocytes. All Finnish LPI patients share the same founder mutation 1181-2A-->T (LPI(Fin)) not found in LPI patients elsewhere. Mutation screening of 20 non-Finnish LPI patients revealed 10 novel mutations: four deletions, two missense mutations, two nonsense mutations, a splice site mutation and a tandem duplication. Five LPI mutations (L334R, G54V, 1291delCTTT, 1548delC and LPI(Fin)) were studied functionally. All mutant proteins failed to co-induce amino acid transport activity when expressed with 4F2hc in Xenopus oocytes. Immunostaining experiments revealed that frameshift mutants 1291delCTTT, 1548delC and LPI(Fin)remained intracellular on expression with 4F2hc. In contrast, the missense mutants L334R and G54V reached the oocyte plasma membrane when co-expressed with 4F2hc, demonstrating that they are transport-inactivating mutations. This finding, together with the strong degree of conservation among all members of this family of amino acid transporters, indicates that residues L334 and G54 play a crucial role in the function of the y(+)LAT-1 transporter.     

High expression of nitric oxide synthases is a favorable prognostic sign in non-small cell lung carcinoma

Immunohistochemical expression of neuronal (n), endothelial (e), and inducible (i) NOS and their association with the type, grade, apoptotic index, proliferation of tumors and the survival of patients were investigated in 89 biopsies of non-small cell lung carcinoma (NSCLC). In tumor cells, expression of iNOS was detected in 35/89 (40%) cases, while 79/89 (89%) and 72/89 (81%) cases showed weak to intense positivity for eNOS and nNOS, respectively. Strong eNOS staining was seen significantly more often in adenocarcinomas than in squamous cells carcinomas (p=0.016), and iNOS immunoreactivity was seen more often in grade I-II tumors than in grade III tumors (p=0.024). There was no significant difference between the low and high apoptotic indexes or between the low and high proliferation rates of tumors in any instance of NOS staining. The patients with tumors showing high nNOS expression tended to have better survival than the others (p=0.06, log-rank; p=0.04, Bresow; p=0.048, Tarone-Ware). Similarly, the patients with tumors showing high expression of iNOS, eNOS and nNOS, as determined by a combined sum index, had a better survival than those with a low sum index for these enzymes (p<0.05). The results show intense expression of eNOS and nNOS, and moderate expression of iNOS in tumor cells of non-small cell carcinoma. Intense NOSs expression seems to be a favorable prognostic sign in non-small cell lung carcinoma.     

Prevalence of shift work disorder among hospital personnel: A cross‐sectional study using objective working hour data

The prevalence of shift work disorder (SWD) has been studied using self‐reported data and the International Classification of Sleep Disorders, Second Edition (ICSD‐2) criteria. We examined the prevalence in relation to ICSD‐2 and ICSD‐3 criteria, work schedules and the number of non‐day shifts (work outside 06:00–18:00 hours) using objective working‐hours data. Secondly, we explored a minimum cut‐off for the occurrence of SWD symptoms. Hospital shift workers without (n = 1,813) and with night shifts (n = 2,917) and permanent night workers (n = 84) answered a survey (response rate 69%) on SWD and fatigue on days off. The prevalence of SWD was calculated for groups with ≥1, ≥3, ≥5 and ≥7 monthly non‐day shifts utilizing the working hours registry. ICSD‐3‐based SWD prevalence was 2.5%–3.7% (shift workers without nights), 2.6%–9.5% (shift workers with nights) and 6.0% (permanent night workers), depending on the cut‐off of non‐day shifts (≥7–1/month, respectively). The ICSD‐2‐based prevalence was higher: 7.1%–9.2%, 5.6%–33.5% and 16.7%, respectively. The prevalence was significantly higher among shift workers with than those without nights (p‐values <.001) when using the cut‐offs of ≥1–3 non‐day shifts. Shift workers with nights who had ≥3 days with ICSD‐3‐based SWD symptoms/month more commonly had fatigue on days off (49.3%) than those below the cut‐off (35.8%, p < .05). The ICSD‐3 criteria provided lower estimates for SWD prevalence than ISCD‐2 criteria, similarly to exclusion of employees with the fewest non‐day shifts. The results suggest that a plausible cut‐off for days with ICSD‐3‐based SWD symptoms is ≥3/month, resulting in 3%–6% prevalence of SWD.     

Genome-wide scan of obesity in Finnish sibpairs reveals linkage to chromosome Xq24

Obesity is a multifactorial trait with evidence of a genetic component. Obesity is very common in all westernized countries, including Finland, where 10% of the adult population has a body mass index of 32 kg/m2 or more. Here we report results from a three-stage genome-wide scan of obesity in 188 affected subjects (body mass index, > or =32 kg/m2) from 87 Finnish families. Initially, 374 markers with an average density of 10 centimorgans were genotyped. The strongest evidence for linkage to obesity was detected on chromosome Xq24, with the marker DXS6804 providing a maximum likelihood score (MLS) 3.14 in a model-free 2-point sibpair analysis. Fine-mapping in an extended sample set of 367 affected subjects from 166 families yielded a multipoint MLS of 3.48 over this X-chromosomal region. The Xq24 region contains a plausible candidate gene, serotonin 2C receptor, variants of which have been shown to predispose to obesity and type II diabetes in mice. Another chromosomal region also provided suggestive evidence of linkage, an area on 18q21, flanking the melanocortin-4 receptor, where a 2-point MLS of 2.42 with marker D18S1155 was obtained with a set of 367 affected subjects. In conclusion, our results in this Finnish study sample suggest that a locus on chromosome Xq24 influences the risk of obesity.     

The effects of 28-day early-life exposure to triphenyl phosphate (TPhP) on odor preference and sexual behavior in female rats

Many chemical substances are detectable in house dust, and they are consequently taken into our bodies via the mouth and nose. Triphenyl phosphate (TPhP), a flame retardant that has an estrogen-like effect in vitro, is present in house dust at high concentrations. Estrogen exposure during development has significant influences on reproductive behavior in rodents, and its effects persist until maturity. In the present study, we investigated the effect of early life exposure to TPhP on the reproductive behavior of female rats. Oral treatment with TPhP (25 or 250 mg/kg), ethinyl estradiol (EE; 15 μg/kg) as a positive control, or sesame oil as a negative control, were given to female rats (from birth to 28 days of age). The 8-week-old rats were bilaterally ovariectomized. At 12–15 weeks of age, the rats were subjected to odor preference and sexual behavior tests. In the odor preference test, the oil group showed significantly higher preference for male odor than female odor, but the low-dose TPhP treatment group lost the preference for male odor, indicating a possible outcome of early life TPhP exposure on sexual recognition. In the sexual behavior test, both the EE and TPhP treatment groups displayed significantly less proceptive behavior. These results suggest that early life exposure to TPhP disturbs the normal sexual behavior of female rats.     

Activated microglia in a rat stroke model express NG2 proteoglycan in peri‐infarct tissue through the involvement of TGF‐β1

We investigated activated microglia in ischemic brain lesions from rats that had been subjected to transient middle cerebral artery occlusion. Activated microglia expressing NG2 chondroitin sulfate proteoglycan (NG2) were found only in the narrow zone (demarcation zone) that demarcated the peri‐infarct tissue and ischemic core. NG2^? activated microglia were abundantly distributed in the peri‐infarct tissue outside the demarcation zone. NG2⁺ microglia but not NG2^? microglia expressed both CD68 and a triggering receptor expressed on myeloid cells 2 (TREM‐2), suggesting that NG2⁺ microglia eliminated apoptotic neurons. In fact, NG2⁺ microglia often attached to degenerating neurons and sometimes internalized NeuN⁺ or neurofilament protein⁺ material. Kinetic studies using quantitative real‐time RT‐PCR revealed that expression of transforming growth factor‐β1 (TGF‐β1) was most evident in the ischemic core; with this marker produced mainly by macrophages located in this region. TGF‐β receptor mRNA expression peaked at 3 days post reperfusion (dpr) in the peri‐infarct tissue, including the demarcation zone. Primary cultured rat microglia also expressed the receptor mRNA. In response to TGF‐β1, primary microglia enhanced the expression of NG2 protein and TREM‐2 mRNA as well as migratory activity. A TGF‐β1 inhibitor, SB525334, abolished these effects. The present results suggest that TGF‐β1 produced in the ischemic core diffused toward the peri‐infarct tissue, driving activated microglial cells to eliminate degenerating neurons. Appropriate control of NG2⁺ microglia in the demarcation zone might be a novel target for the suppression of secondary neurodegeneration in the peri‐infarct tissue. GLIA 2014;62:185–198