Naphthylphthalamic acid associates with and inhibits PIN auxin transporters.

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

Rtr1 is required for Rpb1-Rpb2 assembly of RNAPII and prevents their cytoplasmic clump formation.

RNA polymerase II (RNAPII) is an essential machinery for catalyzing mRNA synthesis and controlling cell fate in eukaryotes. Although the structure and function of RNAPII have been relatively defined, the molecular mechanism of its assembly process is not clear. The identification and functional analysis of assembly factors will provide new understanding to transcription regulation. In this study, we identify that RTR1, a known transcription regulator, is a new multicopy genetic suppressor of mutants of assembly factors Gpn3, Gpn2, and Rba50. We demonstrate that Rtr1 is directly required to assemble the two largest subunits of RNAPII by coordinating with Gpn3 and Npa3. Deletion of RTR1 leads to cytoplasmic clumping of RNAPII subunit and multiple copies of RTR1 can inhibit the formation of cytoplasmic clump of RNAPII subunit in gpn3-9 mutant, indicating a new layer function of Rtr1 in checking proper assembly of RNAPII. In addition, we find that disrupted activity of Rtr1 phosphatase does not trigger the formation of cytoplasmic clump of RNAPII subunit in a catalytically inactive mutant of RTR1. Based on these results, we conclude that Rtr1 cooperates with Gpn3 and Npa3 to assemble RNAPII core.

An Overview of the Pathophysiological Mechanisms of 3-Nitropropionic Acid (3-NPA) as a Neurotoxin in a Huntington's Disease Model and Its Relevance to Drug Discovery and Development.

Animal models are used to better understand the various mechanisms involved in the pathogenesis of diseases and explore potential pathways that will aid in discovering therapeutic targets. 3-Nitropropionic Acid (3-NPA) is a neurotoxin used to induce Huntington's disease (HD)-like symptoms in experimental animals. The 3-NPA is a fungus toxin that impairs the complex II (succinate dehydrogenase) activity of the mitochondria and reduces ATP synthesis, leading to excessive production of free radicals resulting in the degeneration of GABAergic medium spiny neurons (MSNs) in the striatum. This is characterized by motor impairments a key clinical manifestation of HD. 3-NPA has the potential to alter several cellular processes, including mitochondrial functions, oxidative stress, apoptosis, and neuroinflammation mimicking HD-like pathogenic conditions in animals. This review strives to provide a new insight towards the 3-NPA induced molecular dysfunctioning in developing an animal model of HD. Moreover, we summarise several preclinical studies that support the use of the 3-NPA-induced models for drug discovery and development in HD. This review is a collection of various articles that were published from 1977 to 2022 on Pubmed (1639), Web of Science (2139), and Scopus (2681), which are related to the 3-NPA induced animal model.

Classification and Gene Structure of Aquaporins.

Aquaporins (AQPs) are a family of membrane water channels that basically function as regulators of intracellular and intercellular water flow. To date, 13 AQPs, distributed widely in specific cell types in various organs and tissues, have been characterized in humans. A pair of NPA boxes forming a pore is highly conserved among all aquaporins and is also key residues for the classification of AQP superfamily into four groups according to primary sequences. AQPs may also be classified based on their transport properties. So far, chromosome localization and gene structure of 13 human AQPs have been identified, which is definitely helpful for studying phenotypes and potential targets in naturally occurring and synthetic mutations in human or cells.

Evolutionary Overview of Aquaporin Superfamily.

Aquaporins (AQPs) are present not only in three domains of life, bacteria, eukaryotes, and archaea, but also in viruses. With the accumulating arrays of AQP superfamily, the evolutional relationship has attracted much attention with multiple publications on "the genome-wide identification and phylogenetic analysis" of AQP superfamily. A pair of NPA boxes forming a pore is highly conserved throughout the evolution and renders key residues for the classification of AQP superfamily into four groups: AQP1-like, AQP3-like, AQP8-like, and AQP11-like. The complexity of AQP family has mostly been achieved in nematodes and subsequent evolution has been directed toward increasing the number of AQPs through whole-genome duplications (WGDs) to extend the tissue specific expression and regulation. The discovery of the intracellular AQP (iAQP: AQP8-like and AQP11-like) and substrate transports by the plasma membrane AQP (pAQP: AQP1-like and AQP3-like) have accelerated the AQP research much more toward the transport of substrates with complex profiles. This evolutionary overview based on a simple classification of AQPs into four subfamilies will provide putative structural, functional, and localization information and insights into the role of AQP as well as clues to understand the complex diversity of AQP superfamily.

Pharmacophore Oriented MP2 Characterization of Charge Distribution for Anti-SARS-CoV-2 Inhibitor Nirmatrelvir.

Quantum mechanical second order Møller-Plesset (MP2) perturbation theory and density functional theory (DFT) Becke, 3-parameter, Lee-Yang-Parr (B3LYP) and Minnesota 2006 local functional (M06L) calculations were performed to optimize structure of nirmatrelvir and compute the Merz-Kollman electrostatic potential (MK ESP), natural population analysis (NPA), Hirshfeld, charge model 5 (CM5), and mulliken partial charges. The mulliken partial charge distribution of nirmatrelvir exhibits a poor correlation with the MK ESP charges in MP2, B3LYP, and M06L calculations respectively. The NPA, Hirshfeld, and CM5 partial charge scheme of nirmatrelvir indicate a reasonable correlation with MK ESP charge assignments in B3LYP and M06L calculations. The above correlations were not improved by the inclusion of implicit solvation model. The MK ESP and CM5 partial charges show a strong correlation between the results of MP2 and two DFT methods. The three optimized structures present a certain degree of differences from the crystal bioactive conformation of nirmatrelvir, suggesting the nirmatrelvir-enzyme complex is formed in the induced-fit model. The Reactivity of warhead electrophilic nitrile is justified by the relatively weaker strength of π bonds in the MP2 calculations. The nirmatrelvir hydrogen bond acceptors consistently show strong delocalization of lone pair electrons in three calculations, whereas hydrogen bond donors are found to have high polarization on the heavy nitrogen atoms in MP2 computations. This work helps to parametrize the force field of nirmatrelvir and improve accuracy of molecular docking and rational inhibitor design.

Synthetic negative genome screen of the GPN-loop GTPase NPA3 in Saccharomyces cerevisiae.

The GPN-loop GTPase Npa3 is encoded by an essential gene in the yeast Saccharomyces cerevisiae. Npa3 plays a critical role in the assembly and nuclear accumulation of RNA polymerase II (RNAPII), a function that may explain its essentiality. Genetic interactions describe the extent to which a mutation in a particular gene affects a specific phenotype when co-occurring with an alteration in a second gene. Discovering synthetic negative genetic interactions has long been used as a tool to delineate the functional relatedness between pairs of genes participating in common or compensatory biological pathways. Previously, our group showed that nuclear targeting and transcriptional activity of RNAPII were unaffected in cells expressing exclusively a C-terminal truncated mutant version of Npa3 (npa3∆C) lacking the last 106 residues naturally absent from the single GPN protein in Archaea, but universally conserved in all Npa3 orthologs of eukaryotes. To gain insight into novel cellular functions for Npa3, we performed here a genome-wide Synthetic Genetic Array (SGA) study coupled to bulk fluorescence monitoring to identify negative genetic interactions of NPA3 by crossing an npa3∆C strain with a 4,389 nonessential gene-deletion collection. This genetic screen revealed previously unknown synthetic negative interactions between NPA3 and 15 genes. Our results revealed that the Npa3 C-terminal tail extension regulates the participation of this essential GTPase in previously unknown biological processes related to mitochondrial homeostasis and ribosome biogenesis.

A pioneer study on human 3-nitropropionic acid intoxication: Contributions from metabolomics.

The neurotoxin 3-nitropropionic acid (3-NPA) is an inhibitor of succinate dehydrogenase, an enzyme participating both in the citric acid cycle and the mitochondrial respiratory chain. In human intoxications, it produces symptoms such as vomiting and stomach ache in mild cases, and dystonia, coma, and sometimes death in severe cases. We report the results from a liquid chromatography-Orbitrap mass spectrometry metabolomics study mapping the metabolic impacts of 3-NPA intoxication in plasma, urine, and cerebrospinal fluid (CSF) samples of a Norwegian boy initially suspected to suffer from a mitochondrial disease. In addition to the identification of 3-NPA, our findings included a large number of annotated/identified altered metabolites (80, 160, and 62 in plasma, urine, and CSF samples, respectively) belonging to different compound classes, for example, amino acids, fatty acids, and purines and pyrimidines. Our findings indicated protective mechanisms to attenuate the toxic effects of 3-NPA (e.g., decreased oleamide), occurrence of increased oxidative stress in the patient (such as increased free fatty acids and hypoxanthine) and energy turbulence caused by the intoxication (e.g., increased succinate). To our knowledge, this is the first case of 3-NPA intoxication reported in Norway and the first published metabolomics study of human 3-NPA intoxication worldwide. The unexpected identification of 3-NPA illustrates the importance for health care providers to consider intake-related intoxications during diagnostic evaluations, treatment and follow-up examinations for neurotoxicity and a wide range of metabolic derangements.

The mechanism of ß-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation.

ß-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

Npa3 interacts with Gpn3 and assembly factor Rba50 for RNA polymerase II biogenesis.

RNA polymerase II is one of the most vital macromolecular complexes in eukaryotes and the assembly of such complete enzyme requires many factors. Three members of GPN-loop GTPase family Npa3/Gpn1, Gpn2, and Gpn3 participate in the biogenesis of RNA polymerase II with nonredundant roles. We show here that rapid degradation of each GPN protein in yeast leads to cytoplasmic accumulation of Rpb1 and defects in the assembly of RNA polymerase II, suggesting conserved functions of GPN paralogs for RNA polymerase II biogenesis as in humans. Taking advantage of a multicopy genetic screening, we identified GPN3 and assembly factor RBA50 among others as strong suppressors of npa3ts mutants. We further demonstrated that Npa3 interacts with Gpn3 and Rba50, similarly human Gpn1 physically interacts with Gpn3 and RPAP1 (human analog of Rba50). Moreover, a mutual dependency of protein levels of Npa3 and Gpn3 was also clearly presented in yeast using an auxin-inducible degron (AID) system. Interestingly, Rpb2, the second largest subunit of RNA polymerase II was determined to be the subunit that interacts with both Gpn1 and Rba50, indicating a close association of Npa3 and Rba50 in Rpb2 subcomplex assembly. Based on these results, we conclude that Npa3 interacts with Gpn3 and Rba50, for RNA polymerase II biogenesis. We therefore propose that multiple factors may coordinate through conserved regulatory mechanisms in the assembly of RNA polymerase complex.

Contribution and Acceptability of Bacteriological Collection Tools in the Diagnosis of Tuberculosis in Children Infected with HIV.

OBJECTIVE: The objective of this study is to evaluate the feasibility and tolerability of new bacteriological samples to diagnose tuberculosis (TB) in HIV-infected children. METHOD AND PATIENTS: HIV1-infected children with suspicion of TB in Universitary Hospital Sourô Sanon (Burkina Faso) were included in a prospective cohort study. Children underwent three gastric aspirates (GA) if aged <4 years; two GA, one string test (ST) if aged 4-9 years and three sputum, one ST if aged 10-13 years. All children underwent one nasopharyngeal aspirate (NPA) and one stool sample. To assess feasibility and tolerability of procedures, adverse events were identified and pain was rated on different scales. Samples were tested by microscopy, culture, GeneXpert® (Xpert®). RESULTS: Sixty-three patients were included. Mean age was 8.92 years, 52.38% were females. Ninety-five GA, 67 sputum, 62 NPA, 60 stool and 55 ST had been performed. During sampling, the main adverse events were cough at 68/95 GA and 48/62 NPA; sneeze at 50/95 GA and 38/62 NPA and vomiting at 4/55 ST. On the behavioral scale, the average pain score during collection was 6.38/10 for GA; 7.70/10 for NPA and 1.03/10 for ST. Of the 31 cases of TB, bacteriological confirmation was made in 12 patients. CONCLUSION: ST, stool is well-tolerated alternatives specimens for diagnosing TB in children. NPA has a poor feasibility and tolerability in children.

NPA-Cu2+ Complex as a Fluorescent Sensing Platform for the Selective and Sensitive Detection of Glyphosate.

Glyphosate is a highly effective, low-toxicity, broad-spectrum herbicide, which is extensively used in global agriculture to control weeds and vegetation. However, glyphosate has become a potential threat to human and ecosystem because of its excessive usage and its bio-concentration in soil and water. Herein, a novel turn-on fluorescent probe, N-n-butyl-4-(3-pyridin)ylmethylidenehydrazine-1,8-naphthalimide (NPA), is proposed. It efficiently detected Cu2+ within the limit of detection (LOD) of 0.21 µM and displayed a dramatic turn-off fluorescence response in CH3CN. NPA-Cu2+ complex was employed to selectively and sensitively monitor glyphosate concentrations in real samples accompanied by a fluorescence turn-on mode. A good linear relationship between NPA and Cu2+ of glyphosate was found in the range of 10-100 µM with an LOD of 1.87 µM. Glyphosate exhibited a stronger chelation with Cu2+ than NPA and the system released free NPA through competitive coordination. The proposed method demonstrates great potential in quantitatively detecting glyphosate in tap water, local water from Songhua River, soil, rice, millet, maize, soybean, mung bean, and milk with mild conditions, and is a simple procedure with obvious consequences and no need for large instruments or pretreatment.

Apolipoprotein D-mediated preservation of lysosomal function promotes cell survival and delays motor impairment in Niemann-Pick type A disease.

Lysosomal Storage Diseases (LSD) are genetic diseases causing systemic and nervous system dysfunction. The glia-derived lipid binding protein Apolipoprotein D (ApoD) is required for lysosomal functional integrity in glial and neuronal cells, ensuring cell survival upon oxidative stress or injury. Here we test whether ApoD counteracts the pathogenic consequences of a LSD, Niemann Pick-type-A disease (NPA), where mutations in the acid sphingomyelinase gene result in sphingomyelin accumulation, lysosomal permeabilization and early-onset neurodegeneration. We performed a multivariable analysis of behavioral, cellular and molecular outputs in 12 and 24 week-old male and female NPA model mice, combined with ApoD loss-of-function mutation. Lack of ApoD in NPA mice accelerates cerebellar-dependent motor deficits, enhancing loss of Purkinje neurons. We studied ApoD expression in brain sections from a NPA patient and age-matched control, and the functional consequences of ApoD supplementation in primary human fibroblasts from two independent NPA patients and two control subjects. Cell viability, lipid peroxidation, and lysosomal functional integrity (pH, Cathepsin B activity, Galectin-3 exclusion) were examined. ApoD is endogenously overexpressed in NPA patients and NPA mouse brains and targeted to lysosomes of NPA patient cells, including Purkinje neurons and cultured fibroblasts. The accelerated lysosomal targeting of ApoD by oxidative stress is hindered in NPA fibroblasts, contributing to NPA lysosomes vulnerability. Exogenously added ApoD reduces NPA-prompted lysosomal permeabilization and alkalinization, reverts lipid peroxides accumulation, and significantly increases NPA cell survival. ApoD administered simultaneously to sphingomyelin overload results in complete rescue of cell survival. Our results reveal that ApoD protection of lysosomal integrity counteracts NPA pathology. ApoD supplementation could significantly delay not only the progression of NPA disease, but also of other LSDs through its beneficial effects in lysosomal functional maintenance.

High-flow nasal cannula improves clinical efficacy of airway management in patients undergoing awake craniotomy.

BACKGROUND: Awake craniotomy requires specific sedation procedure in an awake patient who should be able to cooperate during the intraoperative neurological assessment. Currently, limited number of literatures on the application of high-flow nasal cannula (HFNC) in the anesthetic management for awake craniotomy has been reported. Hence, we carried out a prospective study to assess the safety and efficacy of humidified high-flow nasal cannula (HFNC) airway management in the patients undergoing awake craniotomy. METHODS: Sixty-five patients who underwent awake craniotomy were randomly assigned to use HFNC with oxygen flow rate at 40 L/min or 60 L/min, or nasopharynx airway (NPA) device in the anesthetic management. Data regarding airway management, intraoperative blood gas analysis, intracranial pressure, gastric antral volume, and adverse events were collected and analyzed. RESULTS: Patients using HFNC with oxygen flow rate at 40 or 60 L/min presented less airway obstruction and injuries. Patients with HFNC 60 L/min maintained longer awake time than the patients with NPA. While the intraoperative PaO2 and SPO2 were not significantly different between the HFNC and NPA groups, HFNC patients achieved higher PaO2/FiO2 than patients with NPA. There were no differences in Brain Relaxation Score and gastric antral volume among the three groups as well as before and after operation in any of the three groups. CONCLUSION: HFNC was safe and effective for the patients during awake craniotomy. TRIAL REGISTRATION: Chinese Clinical Trial Registry, CHiCTR1800016621 . Date of Registration: 12 June 2018.

Quality Outcomes Database Spine Care Project 2012-2020: milestones achieved in a collaborative North American outcomes registry to advance value-based spine care and evolution to the American Spine Registry.

The Quality Outcomes Database (QOD), formerly known as the National Neurosurgery Quality Outcomes Database (N2QOD), was established by the NeuroPoint Alliance (NPA) in collaboration with relevant national stakeholders and experts. The overarching goal of this project was to develop a centralized, nationally coordinated effort to allow individual surgeons and practice groups to collect, measure, and analyze practice patterns and neurosurgical outcomes. Specific objectives of this registry program were as follows: "1) to establish risk-adjusted national benchmarks for both the safety and effectiveness of neurosurgical procedures, 2) to allow practice groups and hospitals to analyze their individual morbidity and clinical outcomes in real time, 3) to generate both quality and efficiency data to support claims made to public and private payers and objectively demonstrate the value of care to other stakeholders, 4) to demonstrate the comparative effectiveness of neurosurgical and spine procedures, 5) to develop sophisticated 'risk models' to determine which subpopulations of patients are most likely to benefit from specific surgical interventions, and 6) to facilitate essential multicenter trials and other cooperative clinical studies." The NPA has launched several neurosurgical specialty modules in the QOD program in the 7 years since its inception including lumbar spine, cervical spine, and spinal deformity and cerebrovascular and intracranial tumor. The QOD Spine modules, which are the primary subject of this paper, have evolved into the largest North American spine registries yet created and have resulted in unprecedented cooperative activities within our specialty and among affiliated spine care practitioners. Herein, the authors discuss the experience of QOD Spine programs to date, with a brief description of their inception, some of the key achievements and milestones, as well as the recent transition of the spine modules to the American Spine Registry (ASR), a collaboration between the American Association of Neurological Surgeons and the American Academy of Orthopaedic Surgeons (AAOS).

Current state and prospects of biotechnology in Central and Eastern European countries. Part II: new and preaccession EU countries(CRO, RO, B&H, SRB).

Innovation holds the potential for economic prosperity. Biotechnology (BT) has proved to be a viable vehicle for the development and utilization of technologies, which has brought not only advances to society, but also career opportunities to nation-states that have enabling conditions. In this review, we assess the current state of BT-related activities within selected new and preaccession EU countries (NPA) of CEE region namely Croatia, Romania, Bosnia and Herzegovina and Serbia, examining educational programs, research activity, enterprises, and the financing systems. The field of BT covers a broad area of activities, including medical, food and agriculture, aquaculture or marine, environmental, biofuels, bioinformatics, and many others. Under the European Commission (EC), member-states are to set their Research and Innovation Strategies for Smart Specialization (RIS3), to identify priorities or strengths in order to develop knowledge intensive economies. As the four countries highlighted in this review are in the early stages of implementing RIS3 or have not yet fully formulated, it presents an opportunity to learn from the successes and failures of those that have already received major structural funds from the EC. A critical point will be the ability of the public and private sectors' actors to align, in the implementation of RIS3 as new investment instruments emerge, and to concentrate efforts on a few select target goals, rather than distribute funding widely without respect to a long-term vision.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Naphthylphthalamic acid and the mechanism of polar auxin transport.

Our current understanding of how plants move auxin through their tissues is largely built on the use of polar auxin transporter inhibitors. Although the most important proteins that mediate auxin transport and its regulation have probably all been identified and the mapping of their interactions is well underway, mechanistically we are still surprisingly far away from understanding how auxin is transported. Such an understanding will only emerge after new data are placed in the context of the wealth of physiological data on which they are founded. This review will look back over the use of a key inhibitor called naphthylphthalamic acid (NPA) and outline its contribution to our understanding of the molecular mechanisms of polar auxin transport, before proceeding to speculate on how its use is likely still to be informative.

In vitro expression and functional characterization of NPA motifs in aquaporins of Nosema bombycis.

Nosema bombycis contains functional aquaporins (NbAQPs), which are key targets for exploring the mechanism of N. bombycis infection; however, the regulation of these NbAQPs remains unknown. The two highly conserved asparagine-proline-alanine sequences (NPA motifs) play important roles in AQP biogenesis. As part of this study, we constructed a series of NbAQP mutants (NbAQP_NPA1, NbAQP_NPA2, and NbAQP_NPA1,2) and expressed them in BmN cells. The results showed that mutations in either NPA motif or in both NPA motifs did not affect NbAQP expression in vitro. After expression in Xenopus laevis oocytes, those injected with wild-type NbAQP rapidly expanded, whereas oocytes injected with NbAQP_NPAs did not significantly change in size. The associated water permeability (pf) of NbAQP_NPAs was significantly reduced five-six times compared to that of wild-type NbAQP. These results indicated that NPA motifs are necessary for the water channel function of AQPs in N. bombycis. The present study shows for the first time that the NbAQP NPA motif has an impact on the water permeability of aquaporin in N. bombycis, thereby providing a platform for further research into the mechanisms underlying the regulation of NbAQP expression.

Molecular cloning and expression analysis of major intrinsic protein gene in Chlamydomonas sp. ICE-L from Antarctica.

Major intrinsic proteins (MIPs) form channels facilitating the passive transport of water and other small polar molecules across membranes. In this study, the complete open reading frame (ORF) of CiMIP1 (GenBank ID KY316061) encoding one kind of MIPs in the Antarctic ice microalga Chlamydomonas sp. ICE-L is successfully cloned using RACE. In addition, the expression patterns of CiMIP1 gene under different conditions of temperature and salinity are determined by qRT-PCR. The ORF of CiMIP1 gene encodes 308 amino acids, and the deduced amino acid sequence shows 74% homology with Chlamydomonas reinhardtii CrMIP1 (GenBank number 159471952). Phylogenetic analysis reveals that algal MIPs are divided into seven groups, and it is speculated that CiMIP1 most likely belongs to the MIPD subfamily. In addition, we are surprised to find that a third NPA motif exists at the carboxy terminus of the target protein except for two highly conserved ones. Expression analysis shows that the transcriptional levels of CiMIP1 gene are upregulated under either lower temperature or higher temperature and high salinity. In summary, the results together have provide new insights into the newly discovered gene in green algae and lay the foundation for further studies on the adaptation mechanism of Chlamydomonas sp. ICE-L to abiotic stresses.