Familial hypobetalipoproteinemia (FHBL), an autosomal co-dominant disorder, is associated with reduced plasma concentrations (<5th percentile for age and sex) of apolipoprotein (apo) B and ␤-migrating lipoproteins. To date, only mutations in APOB encoding prematurely truncated apoB have been found in FHBL. We discovered a novel APOB gene mutation, namely R463W, in an extended Christian Lebanese FHBL kindred. Heterozygotes for R463W had the typical FHBL phenotype, whereas homozygotes had barely detectable apoB-100. The effect of the R463W mutation on apoB secretion was examined using transfected McA-RH7777 cells that expressed one of two recombinant human apoBs, namely B48 and B17. In both cases, the mutant proteins (B48RW and B17RW) were retained within the endoplasmic reticulum and were secreted poorly compared with their wild-type counterparts. Pulse-chase analysis showed that secretion efficiencies of B48RW and B17RW were, respectively, 45 and 40% lower than those of the wild-types. Substitution of Arg 463 with Ala in apoB-17 (B17RA) decreased secretion efficiency by ϳ50%, but substitution with Lys (B17RK) had no effect on secretion, indicating that the positive charge was important. Molecular modeling of apoB predicted that Arg 463 was in close proximity to Glu 756 and Asp 456 . Substitution of Glu 756 with Gln (B17EQ) had no effect on secretion, but substitution of Asp 456 with Asn (B17DN) decreased secretion to the same extent as B17RW. In co-transfection experiments, the mutant B17RW showed increased binding to microsomal triglyceride transfer protein as compared with wild-type B17. Thus, the naturally occurring R463W mutant reveals a key local domain governing assembly and secretion of apoBcontaining lipoproteins. Apolipoprotein (apo)1 B is essential for the formation of triglyceride-rich lipoproteins, namely very low density lipoproteins (VLDL) and chylomicrons (1). In humans, the liver secretes full-length apoB-100 containing 4536 amino acids, whereas the intestine secretes apoB-48 consisting of the aminoterminal 48% of apoB-100 (2). Both forms of apoB are encoded by the APOB gene on chromosome 2, which spans 43 kb and contains 29 exons coding for a 14-kb mRNA (3, 4). ApoB-48 arises from a unique editing process in which cytosine at nucleotide position 6666 is converted to uracil, thereby generating an in-frame stop codon (5). The rat liver produces both apoB-100 and apoB-48, and both forms can assemble VLDL (6).A pentapartite model for apoB-100 on low density lipoproteins (LDL) has been proposed, in which the apoB polypeptide can be divided into five structurally distinct domains, namely NH 2 -␤␣1-␤1-␣2-␤2-␣3-COOH (7). The amino acid sequence of the ␤␣1 domain is homologous to lamprey lipovitellin and microsomal triglyceride transfer protein (MTP) (8,9). The ␤␣1 domain of human apoB has thus been modeled on the basis of the solved lipovitellin structure, in which 13 ␤-strands (amino acids 21-263) form a ␤ barrel, followed by a two-layered helical bundle consisting of 17 ␣-helices (amino acids 294 -592...

Background Delayed cerebral ischemia remains a common and profound risk factor for poor outcome after subarachnoid hemorrhage (SAH). The aim of our current study is to define the role of endothelial nitric oxide synthase (eNOS) in isoflurane conditioning‐induced neurovascular protection after SAH. Methods and Results Ten‐ to 14‐week‐old male wild‐type mice (C57BL/6) as controls and eNOS knockout male mice (strain # 002684) were obtained for the study. Animals underwent either sham surgery, SAH surgery, or SAH with isoflurane conditioning. Anesthetic post conditioning was performed with isoflurane 2% for 1 hour, 1 hour after SAH. Normothermia was maintained with the homeothermic blanket. In a separate cohort, nitric oxide synthase was inhibited by a pan nitric oxide synthase inhibitor, L‐nitroarginine methyl ester. Vasospasm measurement was assessed 72 hours after SAH and neurological function was assessed daily. Isoflurane‐induced changes in the eNOS protein expression were measured. eNOS protein expression was significantly increased by isoflurane conditioning in naïve mice as well as mice subjected to SAH. Vasospasm of the middle cerebral artery and neurological deficits were evident following SAH versus sham surgery, both in wild‐type mice and eNOS knockout mice. Isoflurane conditioning attenuated vasospasm and neurological deficits in wild‐type mice. This delayed cerebral ischemia protection was lost in L‐nitroarginine methyl ester ‐administered mice and eNOS knockout mice. Conclusions Our data indicate isoflurane conditioning provides robust protection against SAH‐induced vasospasm and neurological deficits, and that this delayed cerebral ischemia protection is critically mediated via isoflurane‐induced augmentation of eNOS.

Aneurysmal subarachnoid hemorrhage is a devastating condition causing significant morbidity and mortality. While outcomes from subarachnoid hemorrhage have improved in recent years, there continues to be significant interest in identifying therapeutic targets for this disease. In particular, there has been a shift in emphasis toward secondary brain injury that develops in the first 72 hours after subarachnoid hemorrhage. This time period of interest is referred to as the early brain injury period and comprises processes including microcirculatory dysfunction, blood-brain-barrier breakdown, neuroinflammation, cerebral edema, oxidative cascades, and neuronal death. Advances in our understanding of the mechanisms defining the early brain injury period have been accompanied by improved imaging and nonimaging biomarkers for identifying early brain injury, leading to the recognition of an elevated clinical incidence of early brain injury compared with prior estimates. With the frequency, impact, and mechanisms of early brain injury better defined, there is a need to review the literature in this area to guide preclinical and clinical study.

We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N 158 ) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N 158 , N 956 , N 1341 , N 1350 , and N 1496 ), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by Ͼ 50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d Ͻ 1.02 and d Ͼ 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB. The asparagine (N)-linked oligosaccharides of proteins are an important component of the quality control mechanisms of eucaryotic cells. Multiple roles have been assigned to N-linked oligosaccharides, including folding of nascent polypeptides, protection from proteolytic degradation, intracellular trafficking, secretion, cell surface expression, maintenance of protein conformation, and enzymatic activity (1-3). Human apolipoprotein (apo) B-100, a major structural protein of VLDL synthesized in the liver, is a 4,536 amino acid glycoprotein. There are 20 potential N-linked glycosylation sites within apoB-100, of which 16 Asn residues are conjugated with oligosaccharides on plasma LDL. Each mole of apoB-100 contains 5-6 mol of high-mannose type, and 8-10 mol of complex type oligosaccharides (4). As a member of the vitellogenin family of lipid transport and storage proteins (5-7), apoB-100 possesses numerous amphipathic ␣ -helices and ␤ -strands that constitute the major structural framework for the assembly and integrity of triglyceride-rich lipoproteins (8, 9). The precise amino acid sequences within apoB-100 that are involved ...

BACKGROUND Delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (SAH) has been identified as an independent predictor of poor outcome in numerous studies. OBJECTIVE To investigate the potential protective role of inhalational anesthetics against angiographic vasospasm, DCI, and neurologic outcome in SAH patients. METHODS After Institutional Review Board approval, data were collected retrospectively for SAH patients who received general anesthesia for aneurysm repair between January 1st, 2010 and May 31st, 2018. Primary outcomes were angiographic vasospasm, DCI, and neurologic outcome as measured by modified Rankin scale at hospital discharge. Univariate and logistic regression analysis were performed to identify independent predictors of these outcomes. RESULTS The cohort included 390 SAH patients with an average age of 56 ± 15 (mean ± SD). Multivariate logistic regression analysis identified inhalational anesthetic only technique, Hunt-Hess grade, age, anterior circulation aneurysm and average intraoperative mean blood pressure as independent predictors of angiographic vasospasm. Inhalational anesthetic only technique and modified Fishers grade were identified as independent predictors of DCI. No impact on neurological outcome at time of discharge was noted. CONCLUSION Our data provide additional evidence that inhalational anesthetic conditioning in SAH patients affords protection against angiographic vasospasm and new evidence that it exerts a protective effect against DCI. When coupled with similar results from preclinical studies, our data suggest further investigation into the impact of inhalational anesthetic conditioning on SAH patients, including elucidating the most effective dosing regimen, defining the therapeutic window, determining whether a similar protective effect against early brain injury, and on long-term neurological outcome exists.