Effect of Various Pathological Conditions on Nitric Oxide Level and L-Citrulline Uptake in Motor Neuron-Like (NSC-34) Cell Lines.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a non-essential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [14C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [14C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [14C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [14C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

ALS/FTD-associated mutation in cyclin F inhibits ER-Golgi trafficking, inducing ER stress, ERAD and Golgi fragmentation.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.

Modeling of the As (III) adsorption using Fe impregnated polyethylene terephthalate char matrix: A statistical approach.

The present research aimed to analyse the impact of economical Fe impregnated polyethylene terephthalate (PET) char (PETC-Fe) for adsorption of As (III) through series of column experiments. For an inlet arsenite concentration of 1,000 µg/L, PETC-Fe exhibits excellent uptake capacity of 1,892 µg/g. Central composite design (CCD) in response surface methodology (RSM) was used to evaluate the influence of various process variables on the response function (breakthrough time) for optimization and assessment of interaction effects. The breakthrough time is more responsive to influent As (III) concentration and bed height than inlet flow rate, according to the perturbation plot. Adams-Bohart, Bed Depth Service Time (BDST) model, and Thomas models were used to model the dynamics of the adsorption system. The BDST model suited the experimental data well in the early part of the breakthrough curve, but there were minor variations over the breakpoints. Despite the fact that the experimental values and the data sets estimated using the Adams-Bohart model followed a similar pattern, they differed slightly. The PETC-Fe was found to be a sustainable and highly economical adsorbent, with a desorption performance of more than 97%, indicating the adsorbent's reusability. This adsorbent's excellent As (III) uptake capacity and regeneration performance imply that it might be used in industrial/domestic applications, and the information obtained could aid in future scaling up of the adsorption system.

L-Citrulline Level and Transporter Activity Are Altered in Experimental Models of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease caused by the death of the neurons regulating the voluntary muscles which leads to the progressive paralysis. We investigated the difference of transport function of L-citrulline in ALS disease model (NSC-34/hSOD1G93A, MT) and a control model (NSC-34/hSOD1wt, WT). The [14C]L-citrulline uptake was significantly reduced in MT cells as compared with that of control. The Michaelis-Menten constant (Km) for MT cells was 0.67 ± 0.05 mM, whereas it was 1.48 ± 0.21 mM for control. On the other hand, the Vmax values for MT and control were 10.9 ± 0.8 nmol/mg protein/min and 18.3 ± 2.9 nmol/mg protein/min, respectively. The Km and Vmax values showed the high affinity and low capacity for MT as compared with control. Moreover, the uptake of [14C]L-citrulline was significantly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) and harmaline which is the inhibitor of the large neutral amino acid transporter1 (LAT1) in NSC-34 cell lines. Furthermore, [14C]L-citrulline uptakes took place in Na+-independent manner. It was also inhibited by the neutral amino acids such as citrulline and phenylalanine. Likewise, L-dopa, gabapentin, and riluzole significantly inhibited the [14C]L-citrulline uptake. It shows the competitive inhibition for L-dopa in ALS cell lines. On the other hand, [14C]L-citrulline uptake in the presence of riluzole showed competitive inhibition in WT cells, whereas it was uncompetitive for MT cells. The small interfering RNA experiments showed that LAT1 is involved in the [14C]L-citrulline uptake in NSC-34 cell lines. On the other hand, in the examination of the alteration in the expression level of LAT1, it was significantly lower in MT cells as compared with that of control. Similarly, in the spinal cord of ALS, transgenic mice revealed a slight but significant decrease in LAT1 immunoreactivity in motor neurons of ALS mice compared with control. However, the LAT1 immunoreactivity in non-motor neurons and in astrocytes was relatively increased in the spinal cord gray matter of ALS mice. The experimental evidences of our results suggest that the change of transport activity of [14C]L-citrulline may be partially responsible for the pathological alteration in ALS.

Intron 2 substitution resulted in HLA-DQB1*02:01:01:02 variant in a Kashmiri Brahmin individual from North India.

HLA-DQB1*02:01:01:02 differs from HLA-DQB1*02:01:01:01 by one nucleotide change in intron 2 at position 3949 (G>C).

Characterization of the novel HLA-C*07:862 allele in a North Indian hematopoietic stem cell donor.

One nucleotide change (G > C) in the exon 4 of HLA-C*07:02:01:01 results in the novel allele, HLA-C*07:862.

The novel HLA-C*07:02:01:97 allele identified in a Chang tribal individual from Nagaland, North-East India.

Two nucleotide changes in the 3'-UTR of HLA-C*07:02:01:01 results in the novel allele, HLA-C*07:02:01:97.

A novel allele HLA-B*38:02:01:02, identified in an Angami individual from Nagaland, North-East India.

A single nucleotide change (C to T) in HLA-B*38:02:01 results in the novel allele, HLA-B*38:02:01:02.

HLA-C*07:06:01:06-A new HLA-C allele identified in an Ao tribal individual from Nagaland, North-East India.

Two nucleotide changes in the 3'untranslated region of HLA-C*07:06:01:01 results in the novel allele, HLA-C*07:06:01:06.

The HLA-B*13:01:01:03 allele characterized in four individuals from the tribal population of Nagaland, North-East India.

A single nucleotide change (G to T) in HLA-B*13:01:01:01 results in the novel allele, HLA-B*13:01:01:03.

Recognition of the novel HLA-A*11:01:01:25 allele in the tribal population of Nagaland, North-East India.

One nucleotide change (G>A) at intron 1 of HLA-A*11:01:01:01 results in the novel allele, HLA-A*11:01:01:25.

HLA-A*33:03:01:06 allele identified in an individual from the Kashmiri Brahmin population of North India.

HLA-A*33:03:01:06 differs from HLA-A*33:03:01:01 by one nucleotide substitution (T- > C) in intron 3.