Maternal immune activation dysregulation of the fetal brain transcriptome and relevance to the pathophysiology of autism spectrum disorder.

Maternal immune activation (MIA) via infection during pregnancy is known to increase risk for autism spectrum disorder (ASD). However, it is unclear how MIA disrupts fetal brain gene expression in ways that may explain this increased risk. Here we examine how MIA dysregulates rat fetal brain gene expression (at a time point analogous to the end of the first trimester of human gestation) in ways relevant to ASD-associated pathophysiology. MIA downregulates expression of ASD-associated genes, with the largest enrichments in genes known to harbor rare highly penetrant mutations. MIA also downregulates expression of many genes also known to be persistently downregulated in the ASD cortex later in life and which are canonically known for roles in affecting prenatally late developmental processes at the synapse. Transcriptional and translational programs that are downstream targets of highly ASD-penetrant FMR1 and CHD8 genes are also heavily affected by MIA. MIA strongly upregulates expression of a large number of genes involved in translation initiation, cell cycle, DNA damage and proteolysis processes that affect multiple key neural developmental functions. Upregulation of translation initiation is common to and preserved in gene network structure with the ASD cortical transcriptome throughout life and has downstream impact on cell cycle processes. The cap-dependent translation initiation gene, EIF4E, is one of the most MIA-dysregulated of all ASD-associated genes and targeted network analyses demonstrate prominent MIA-induced transcriptional dysregulation of mTOR and EIF4E-dependent signaling. This dysregulation of translation initiation via alteration of the Tsc2-mTor-Eif4e axis was further validated across MIA rodent models. MIA may confer increased risk for ASD by dysregulating key aspects of fetal brain gene expression that are highly relevant to pathophysiology affecting ASD.

Factors affecting measurement of specific methanogenic activity.

Specific methane production (SMP) tests have been used to determine the potential loading rate capacity of anaerobic reactors, to characterize biomass prior to its use as an inoculum for new anaerobic reactors, to detect changes in biomass activity during operation, or to assess the occurrence of toxic conditions. SMP tests also provide a basis for estimating specific methanogenic activity in mixed anaerobic cultures. SMP protocols used to date have varied widely in both procedure and objective. Tests conducted by the present authors indicated that biomass concentration, substrate type and concentration, and mixing intensity are factors that can affect the results of SMP tests.

Identification and characterization of novel antimicrobial decapeptides generated by combinatorial chemistry.

Novel combinatorial libraries consisting of simplified amino acid sequences were designed to screen for peptides active against the Candida albicans membrane. A novel decapeptide, KKVVFKVKFK, that had a unique primary amino acid sequence was identified in this work. This peptide irreversibly inhibited the growth of C. albicans and showed a broad range of antibacterial activity but no hemolytic activity. Circular dichroism spectra revealed that the predominant secondary structure of this peptide strongly depended on the membrane-mimetic environments; the peptide preferred to form an amphipathic alpha-helical structure in the presence of 50% trifluoroethanol, while it preferred to adopt a distorted alpha-helical structure in the presence of sodium dodecyl sulfate micelles. Experiments in which dye was released from vesicles indicated that this novel antimicrobial peptide killed microorganisms through the action on the membrane as its primary target. Replacement of amino acids in this active decapeptide on the basis of information from the libraries could provide unique information about factors affecting its antimicrobial activity such as its secondary structure, net positive charge, and hydrophobicity.

Identification and characterization of the antimicrobial peptide corresponding to C-terminal beta-sheet domain of tenecin 1, an antibacterial protein of larvae of Tenebrio molitor.

An active fragment was identified from tenecin 1, an antibacterial protein belonging to the insect defensin family, by synthesizing the peptides corresponding to the three regions of tenecin 1. Only the fragment corresponding to the C-terminal beta-sheet domain showed activity against fungi as well as Gram-positive and Gram-negative bacteria, whereas tenecin 1, the native protein, showed activity only against Gram-positive bacteria. CD spectra indicated that each fragment in a membrane-mimetic environment might adopt a secondary structure corresponding to its region in the protein. The leakage of dye from liposomes induced by this fragment suggested that this fragment acts on the membrane of pathogens as a primary mode of action. A comparison between the structure and the activity of each fragment indicated that a net positive charge was a prerequisite factor for activity. To the best of our knowledge this is the first report in which the fragment corresponding to the beta-sheet region in antibacterial proteins, which consists of alpha-helical and beta-sheet regions, has been identified as a primary active fragment.

Purification and molecular cloning of a platelet aggregation inhibitor from the snake (Agkistrodon halys brevicaudus) venom.

A platelet glycoprotein IIb-IIIa (GP IIb-IIIa) antagonist, salmosin, was purified to homogeneity from Korean snake (Agkistrodon halys brevicaudus) venom by means of chromatographic fractionations. We have isolated the cDNA encoding salmosin by using the cDNA library of the snake venom gland and analyzed its complete nucleotide sequence. The molecular identity was confirmed by comparison of the deduced amino acid sequence with the directly determined primary structure of salmosin. This protein is a single-chain polypeptide composed of 73 amino acids including 12 cysteines as well as the sequence Arg-Gly-Asp, a proposed recognition site of adhesive proteins. The primary sequence of salmosin shows considerable homology to previously described proteins of snake venom GP IIb-IIIa antagonist family. A molecular mass of 7474 for the protein was determined by matrix-assisted laser desorption ionization mass spectrometry. Salmosin inhibits GP IIb-IIIa binding to immobilized fibrinogen with an IC50 of 2.2 nM and ADP-induced platelet aggregation with an IC50 of 131 nM, respectively. This work demonstrates the purification, characterization, and cDNA cloning of salmosin, a platelet aggregation inhibitor that may have therapeutic potential as an antithrombotic agent.

Unusually stable helical kink in the antimicrobial peptide--a derivative of gaegurin.

The structure of an active analog of the antibacterial peptide gaegurin was investigated by CD and NMR spectroscopy. The NOE connectivities showed that 21 out of 24 residues formed an a-helix despite the presence of a central proline. CD and NMR analysis indicates that the helix is in fast equilibrium with random coil. From chemical shift analysis of the amide protons, the distances of hydrogen bonding in the helix were calculated, and manifested obvious periodicity which implied a kink in the middle of the helix. 1D amide proton exchange experiments provided further evidence of an exceptionally stable kink. It is inferred that this kink is important not only to the function of the peptide but also to the early stage of the folding as a nucleation site.

Molecular cloning of cDNAs encoding precursors of frog skin antimicrobial peptides from Rana rugosa.

Gaegurins, a family of peptide antibiotics with sizes ranging from 24 to 37 amino acids, have recently been purified from Rana rugosa skin (Park, J.M., Jung, J.-E. and Lee, B.J. (1994) Biochem. Biophys. Res. Commun. 205, 948-954). Two complete cDNAs encoding gaegurins 4 and 5 were isolated from a library constructed with the frog skin mRNAs. Each clone contained a single open reading frame that encodes a gaegurin precursor polypeptide. The deduced amino acid sequences revealed that the precursors have a unique tripartite structure: a putative signal sequence at the NH2-terminus followed by an acidic spacer region rich in glutamic and aspartic acids, and a mature gaegurin peptide at the COOH-terminus. Similar modes of organization were also found in antimicrobial or opioid peptide precursors of other frog species, although their mature peptides show little sequence homology. The family of peptides with this characteristic now expands. Northern analysis revealed that gaegurins are extensively expressed in the skin tissue, but not in liver and muscle.

Identification of immunodominant epitopes in the core and non-structural region of hepatitis C virus by enzyme immunoassay using synthetic peptides.

Thirty-two synthetic peptides, components of the core and non-structural protein of Hepatitis C virus (HCV), were tested for their reactivities against antibodies in sera of healthy, HCV antibody positive of chronic liver disease patients. Among them, 8 of the core peptides, 4 of the NS4 peptides and 3 of the NS5 peptides reacted with the HCV infected sera. In particular, C22 (core peptide) and NS4-1924 (NS4 peptide) were most reactive with the serum samples giving a positive signal with commercially available enzyme-linked immunosorbent assay (ELISA) kit. Our results indicate that the immunodominant regions of the HCV-derived proteins are located at three regions in the core protein, three regions in the NS4 protein, and one region in the NS5 protein. These results indicate that the selected peptides are useful antigens in detecting antibodies in the sera from individuals infected with HCV.

Step-fortifications of nutrients in mammalian cell culture.

A series of high-density media for mammalian cell culture were developed by step-fortifications of most nutrient components in RPMI-1640 medium. Each medium constituting the series was constructed to meet in vitro cell growth limitations. Four different cell lines were cultivated in the media series, and their growth characteristics were observed. Maximum cell densities varied in the range of 0.4 to 1.3 x 10(7) cells/mL, depending on cell lines. Cell growth responses to each of the media series were analyzed in terms of cell density and cell mass. Step increases of cell mass in the range of 1.3 to 3.7 g/L were observed according to the step-fortifications of nutrients. Also, the characteristics of each cell line were compared in terms of metabolic yields and specific productions of lactic acid and ammonium ion. The effect of step-fortifications of nutrients on the production of monoclonal antibody was also examined. Apparent differences in metabolic characteristics among cell lines were observed. Experimental results suggested that the different cell sizes and metabolic characteristics of each cell line resulted in cell-line-specific responses to the step-fortifications. The significant influence of nutritional fortifications on high-density culture of mammalian cells was evaluated.

Repeated fed-batch culture of hybridoma cells in nutrient-fortified high-density medium.

Long-term high-density cultivation of the hybridoma 2c3.1 was successfully carried out in a repeated fed-batch mode using high-density media that were constructed to meet in vitro cell growth limitations. The high-density culture was possible in a range of 0.5 approximately 1.0 x 10(7) cells/mL in MBRI 40-02 medium for over 2500 h by the repeated supplementation of the most fortified medium, MBRI 40-03, and consequently, distinct enhancement of MAb production was achieved. MAb concentrations were maintained around 1 g/L for about 1000 h of the process and the maximum MAb concentration was around 1.56 g/L. The result supported strongly the fact that the nutritional fortification was the most critical factor for high-density cell culture in vitro. The mean chromosome number of the hybridoma 2c3.1 was maintained stably for about 1500 h, whereas gradual loss of the MAb activity was apparent during the long-term cultivation.

Identification of clones containing DNA complementary to phenylethanolamine N-methyltransferase mRNA.

Specific poly(A)mRNA for phenylethanolamine N-methyltransferase was isolated from bovine adrenal medulla by immunoprecipitation of polysomal mRNA with antibodies to bovine adrenal phenylethanolamine N-methyltransferase. Antibody-polysome complexes were recovered by Protein A Sepharose affinity chromatography. Phenylethanolamine N-methyltransferase mRNA, enriched 50-fold as judged by quantitative immunoprecipitation of translation products, was used as a template for the synthesis of complementary DNA (cDNA). Double-stranded cDNA was tailed with deoxycytosine and inserted into the Pst 1 site of poly(dG)-tailed plasmid pBR322. The resultant recombinant plasmids were used to transform competent E. coli strain 294. Tetracycline-resistant ampicillin-sensitive clones were screened by positive hybridization selection, and preliminary screening identified 2 out of 36 clones containing phenylethanolamine N-methyltransferase cDNA inserts. One phenylethanolamine N-methyltransferase cDNA insert was isolated from the plasmid DNA by digestion with Pst 1 and was found to be approximately 350 base-pairs in length. Northern blot analysis revealed that this phenylethanolamine N-methyltransferase cDNA probe strongly hybridized to an RNA species of approximately 1100 nucleotides.

Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly.

The 200,000-dalton neurofilament subunit (P200) and the 160,000-dalton (P160) and 78,000-dalton (P78) neurofilament subunits were partially purified from bovine brain. Intact neurofilaments were prepared by high-speed and sucrose-zone centrifugation. The crude neurofilament was solubilized in 8 M urea solution containing pyridine, formic acid, and 2-mercaptoethanol. The solubilized neurofilament was purified by carboxymethyl (CM) cellulose column and hydroxylapatite column chromatography. The P200 was purified as separate from P160 and P78, but the P160 and P78 subunits were copurified on CM cellulose, hydroxylapatite, Bio-Gel A150m, and Sephadex G-150 column chromatography. Electron microscopy of these purified neurofilament subunits revealed the P200 subunit as a globular structure, and the P160 and P78 subunits as a rod-shaped structure extending up to 120 nm with a 8- to 12-nm width. In the presence of 200 mM KCl, 15 mM MgCl2, and 1 mM ATP, the purified subunits assembled into long filaments. Under the assembly condition, P160 and P78 subunits elongated up to 500 nm, but the longer filament formation required the presence of P200 subunits. The filaments formed in vitro were of two types: long straight filaments and intertwined knobby-type filaments. From these results, we have suggested that P160 and P78 form the neurofilament backbone structure and P200 facilitates the assembly of the backbone units into longer filaments.

Murine cytomegalovirus-induced protein synthesis.

Murine cytomegalovirus (MCMV)-induced protein synthesis in mouse embryo fibroblast (MEF) cells was studied using polyacrylamide gradient SDS gel electrophoresis and autoradiography. Synthesis of at least 14 virus induced proteins (VIPs) was consistently detected in a lytic cycle. They were designated VIPs 132, 118, 99, 98, 88, 81, 76, 74, 58, 56, 51, 38, 36 and 33 on the basis of their mol. wt. Judging from the pattern of the rate of protein synthesis, VIPs can be classified into three groups: group A VIPs were synthesized actively for a brief period of time and then their synthesis was no longer detectable. This group included two major VIPs, 98 and 88 and three minor VIPs, 58, 56 and 38. Group B VIPs 81, 74, 36 and 33 were similar to group A except that, following a brief period of active synthesis, a low level of synthesis continued during the entire lytic cycle. Group C VIPs 132, 118, 99, 76 and 51 were synthesized at low steady levels at all times after initiation and seemed to accumulate slowly. According to temporal sequences of initiation of VIP synthesis, these proteins can also be divided into three groups: immediate early, early and late VIPs. The synthesis of the immediate early VIPs 132, 98, 88, 81, 76, 74 and 38 was initiated immediately after virus infection. The early VIPs included 58, 56, 51, 36 and 33 and their synthesis was initiated from 1 to 3 h post-infection. VIPs 118, 99 and several minor VIPs were first synthesized during 12 to 13 h post-infection which corresponded to the time of initiation of virus DNA synthesis and they are classified as late VIPs. Cycloheximide reversal experiments indicated that the initiation of synthesis of early VIPs must be preceded by the synthesis of immediate early VIPs. In the presence of actinomycin D, the immediate early VIPs (0 to 1 h post-infection) were not synthesized indicating that immediate early VIPs are translated from virus mRNA synthesized after virus infection.

Inactivation of cytomegalovirus and Semliki Forest virus by butylated hydroxytoluene.

Butylated hydroxytoluene (BHT) is an antioxidant that is widely used in foods because it prevents spoilage by delaying degradation of lipid components. This hydrophobic compound inactivated human and murine cytomegalovirus (CMV) and Semliki Forest virus (SFV). Both human and murine CMV were inactivated more than 90% by 40 microgram of BHT/ml after incubation for 1 hr at 37 C. Under the same conditions, SVF was inactivated about 75%, whereas poliovirus, which does not contain lipid membrane as a part of its structure, was not inactivated at all. Vaccinia virus was less sensitive to BHT than was CMV or SFV.

Complement-fixing antigen of human cytomegaloviruses.

The titer of complement-fixing (CF) antigen of human cytomegalovirus (CMV) strain C87 was not reduced by 10 cycles of freezing and thawing or by storage at 4 C for two months, although the virus was labile at 37 C and 100 C. The rapid increase in titers of CF antigen and plaque-forming units was seen from days 3 through 6 after infection; however, CF antigen was detected 24 hr after infection. The molecular weight of CF antigen was estimated by gel filtration to be greater than 1.5 X 10(7) daltons. Electrophoresis of CF antigens from five strains of human CMV (AD169, C87, Espilant [Esp.], Davis, and Towne) in a gradient polyacrylamide gel slab in the presence of sodium dodecyl sulfate revealed two specific polypeptides with molecular weights of 140,000 and 66,000 daltons, respectively. The smaller polypeptide was glycosylated. Preparations of CF antigen from murine CMV and herpes simplex virus type 1 contained polypeptides of 140,000 and 145,000 daltons, respectively. However, the polypeptide with a molecular weight of 66,000 daltons was seen only in preparations of human CMV.

Analysis of structural polypeptides of purified human cytomegalovirus.

Human cytomegalovirus strain C87 was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 X g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. (ii) After two consecutive isopycnic potassium tartrate gradient centrifugations (20 to 50%), coinciding peaks of plaque titer, protein, and radioactivity were found at a density of from 1.20 to 1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from two mixtures: (i) [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid; (ii) unlabeled extracellular virus mixed with an equal volume of [357a1methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e. about 2.5% of the protein in the purified virus preparation could be accounted for by host protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VPs), and their molecular weights ranged from 11,000 to 290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine labeled virus showed six bands. Four of these were so broad that several VPs corresponded to each of the glycosylated bands. When heavy (two fractions close to 1.21 g/cm3) and light (two fractions close to 1.20 g/cm3) fractions of the PFU peak from the second potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.

Analysis of structural proteins of purified murine cytomegalovirus.

Murine cytomegalovirus propagated in mouse embryo fibroblasts was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 x g for 90 min. (ii) The concentrated virus was passed through a Bio-Rad Bio-Gel A-15m column to eliminate contaminating materials smaller than 15 x 10(6) daltons. Most of the virus was recovered in the void volume of the column. (iii) Two consecutive centrifugations through 20 to 50% potassium tartrate gradients were performed. After the second tartrate gradient centrifugation, symmetrical, coinciding peaks of plaque titer, protein, and radioactivity were found at a density between 1.20 g/cm3 and 1.21 g/cm3. To establish purification criteria, virus was purified from two different mixtures: [35S]methionine-labeled extracellular virus, mixed with an equal volume of unlabeled normal culture fluid, and unlabeled extracellular virus mixed with an equal volume of [35S]methionine-labeled normal culture fluid. At the end of the procedure, the extent of purification, as judged by the ratio of cellular to viral radioactivity was at least 70-fold. Virus proteins were analyzed by electrophoresis on a 5 to 20% gradient polyacrylamide gel slab. After gel electrophoresis,, Coomassie brilliant blue staining profiles and autoradiograms of the purified virus preparations were compared. At least 33 virus structural protein bands were present. The molecular weights of these proteins ranged from 11,500 to 255,000. The sum of the molecular weights of the virus structural proteins was 2,462,000. Autoradiograms obtained from electrophoresis of purified [14C]glucosamine-labeled virus showed that at lease 6 of the 33 viral structural proteins were glycoproteins.

Multiple forms of elongation factor 1 from calf brain.

Heavy and light forms of elongation factor 1 (EF-1) from calf brain have been partially purified. The heterogeneous heavy species (EF-1(H)) with molecular weights of 2.5 x 10(5) to over 1 x 10(6) appears to be a complex or aggregate of the light form of the enzyme (EF-1(L)); the latter has a molecular weight of between 50,000 and 60,000. EF-1(H) but not EF-1(L), contains significant amounts of free and esterified cholesterol. Although EF-1(H) and EF-1(L) are both active in aminoacyl-tRNA binding to ribosomes, EF-1(L) reacts with GTP and aminoacyl-tRNA more efficiently than EF-1(H).