High-performance liquid chromatographic separation of ascorbic acid,erythorbic acid,dehydroascorbic acid,dehydroerythorbic acid,diketogulonic acid,and diketogluconic acid

High-performance liquid chromatography on a Zorbax NH₂ analytical column, with acetonitrile: 0.05 m KH₂PO₄ (75:25, $\text{w}\text{w}$) used as eluant, has allowed the separation, in less than 14 min, of ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid, diketogulonic acid, and diketogluconic acid. Ultraviolet monitoring at 268 nm allows ascorbic acid and erythorbic acid to be detected at the 25-ng level, while refractive index detection monitors the elution of all six compounds. Tyrosine is a good internal standard, being well separated from the other compounds and having an adequate ultraviolet absorption at 268 nm. We have found dithiothreitol to be effective in rapidly reducing dehydroascorbic acid to ascorbic acid, providing the basis for indirectly determining dehydroascorbic acid after its reduction. The potential of this high-performance liquid chromatographic procedure for evaluating the levels of these compounds in orange juice and urine is demonstrated.     

Cerebrospinal fluid,plasma, and urinary MHPG in children

Cerebrospinal fluid (CSF) free MHPG levels for six autistic and four medicated Tourette Syndrome (TS) patients are in the usual adult range, but levels are elevated in two recently medicated TS patients. Plasma free MHPG levels in unmedicated autistic and TS patients are similar to those in normal boys, but are increased in two of seven medicated TS patients. There is a strong association between plasma free MHPG and urinary total MHPG levels in normal boys. Age and total urinary MHPG are positively correlated in autistic children. MHPG appears to be a useful index of noradrenergic function in childhood neuropsychiatric disorders.     

Coordinate Regulation of Choline Acetyltransferase, Tyrosine Hydroxylase, and Neuropeptide mRNAs by Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor in Cultured Sympathetic Neurons

Abstract: The neurotransmitter phenotype switch that occurs in cultures of rat superior cervical ganglion neurons after treatment with leukemia inhibitory factor or ciliary neurotrophic factor is a useful model permitting investigation of the mechanisms of cytokine-mediated differentiation. Recently the actions of leukemia inhibitory factor and ciliary neurotrophic factor have been linked through their interactions with related receptor complexes. Here we compare the effects of these two cytokines on gene expression in sympathetic neuronal cultures and begin to investigate their mechanisms. We report that, as has been shown for leukemia inhibitory factor, ciliary neurotrophic factor regulates peptides and classical transmitters in these cultures at the mRNA level. In addition, we find that the induction of substance P mRNA by these cytokines is rapid, dependent on protein synthesis, and occurs in 40–50% of superior cervical ganglion neurons in dissociated culture.     

Analytical and preparative HPLC of carbohydrates: inositols and oligosaccharides derived from cellulose and pectin

New methods are given for the production of cellodextrins by the TFA-catalyzed hydrolysis of cellulose and for the subsequent analytical and preparative high performance liquid chromatography of these useful oligosaccharides. In addition, recent methods developed in this laboratory for the analytical and preparative HPLC of inositols and pectin oligosaccharides are reviewed.     

Loss of meiosis in Aspergillus

If strictly mitotic asexual fungi lack recombination, the conventional view predicts that they are recent derivatives from older meiotic lineages. We tested this by inferring phylogenetic relationships among closely related meiotic and strictly mitotic taxa with Aspergillus conidial (mitotic) states. Phylogenies were constructed by using DNA sequences from the mitochondrial small ribosomal subunit, the nuclear ribosomal internal transcribed spacers, and the nuclear 5.8S ribosomal gene. Over 920 bp of sequence was analyzed for each taxon. Phylogenetic analysis of both the mitochondrial and nuclear data sets showed at least four clades that possess both meiotic and strictly mitotic taxa. These results support the hypothesis that strictly mitotic lineages arise frequently from more ancient meiotic lineages with Aspergillus conidial states. Many of the strictly mitotic species examined retained characters that may be vestiges of a meiotic state, including the production of sclerotia, sclerotium-like structures, and hulle cells.      

Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons.

We examined the effects of ciliary neurotrophic factor (CNTF) and depolarization, two environmental signals that influence noradrenergic and cholinergic function, on neuropeptide expression by cultured sympathetic neurons. Sciatic nerve extract, a rich source of CNTF, increased levels of vasoactive intestinal peptide (VIP), substance P, and somatostatin severalfold while significantly reducing levels of neuropeptide Y (NPY). No change was observed in the levels of leu-enkephalin (L-Enk). These effects were abolished by immunoprecipitation of CNTF-like molecules from the extract with an antiserum raised against recombinant CNTF, and recombinant CNTF caused changes in neuropeptide levels similar to those of sciatic nerve extract. Alterations in neuropeptide levels by CNTF were dose-dependent, with maximal induction at concentrations of 5-25 ng/ml. Peptide levels were altered after only 3 days of CNTF exposure and continued to change for 14 days. Depolarization of sympathetic neuron cultures with elevated potassium elicited a different spectrum of effects; it increased VIP and NPY content but did not alter substance P, somatostatin, or L-Enk. Depolarization is known to block cholinergic induction in response to heart cell conditioned medium and we found that it blocked the induction of choline acetyltransferase (ChAT) and peptides by recombinant cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF). In contrast, it did not antagonize the effects of CNTF on either ChAT activity or neuropeptide expression. Thus, while CNTF has effects on neurotransmitter properties similar to those previously reported for CDF/LIF, the actions of these two factors are differentially modulated by depolarization, suggesting that the mechanisms of cholinergic and neuropeptide induction for the two factors differ. In addition, in contrast to CDF/LIF, CNTF did not alter levels of ChAT, VIP, substance P, or somatostatin in cultured dorsal root ganglion neurons. These observations indicate that CNTF and depolarization affect the expression of neuropeptides by sympathetic neurons and provide evidence for an overlapping yet distinct spectrum of actions of the two neuronal differentiation factors, CNTF and CDF/LIF.     

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.      

Induction of human monocyte IL-1 mRNA and secretion during anti-CD3 mitogenesis requires two distinct T cell-derived signals.

The T cell signals that regulate the induction of human monocyte IL-1 during primary immune activation were investigated by using anti-CD3 mitogenesis. The induction of monocyte IL-1 alpha and beta mRNA during anti-CD3 mitogenesis was rapid (less than or equal to 1 h) and required the presence of both T cells and anti-CD3. The addition of T cells plus a nonmitogenic anti-CD5 antibody failed to induce IL-1 alpha or beta mRNA, indicating that IL-1 mRNA induction by anti-CD3 required T cell activation. Experiments using double chamber culture wells revealed that the major initial phase of IL-1 alpha and beta mRNA induction (1 to 12 h) required direct cell contact between monocytes and T cells. A subsequent minor late phase (greater than or equal to 12 h) of IL-1 mRNA was induced independently of cell contact in monocytes that received only soluble factors generated during anti-CD3 mitogenesis and was temporally associated with the appearance in culture supernatants of the late phase IL-1-inducing cytokines, IL-2, IFN-gamma, and TNF-alpha. Metabolic inactivation of T cells using paraformaldehyde demonstrated that the ability of T cells to induce IL-1 mRNA via cell contact was acquired only after activation of T cells via solid phase anti-CD3. Furthermore, pretreatment of T cells with the protein synthesis inhibitor emetine had no effect on T cell-mediated induction of monocyte IL-1 mRNA or cell-associated IL-1 alpha and beta, indicating that the expression of the IL-1 inductive signal did not require protein synthesis. Despite their ability to induce monocyte IL-1 alpha and beta mRNA, activated T cells treated with paraformaldehyde or emetine were no longer able to induce monocytes to secrete IL-1 beta into culture supernatants. However, supernatants from purified T cells that were activated with solid-phase anti-CD3 restored the ability of paraformaldehyde or emetine-treated T cells to induce IL-1 secretion. These studies provide evidence that supports a two-signal model of monocyte IL-1 production during primary immune activation. The first signal leads to the induction of monocyte IL-1 mRNA and is mediated by direct contact with activated T cells, and the second signal is provided by soluble T cell factors and results in IL-1 secretion.     

Luteal regression in the normally cycling rat: apoptosis, monocyte chemoattractant protein-1, and inflammatory cell involvement

In hypophysectomized rats, prolactin induces regression of the corpora lutea. Luteal regression is accompanied by infiltration of monocytes/macrophages, declines in luteal mass and plasma progestins, and increased staining for monocyte chemoattractant protein-1 (MCP-1). We investigated whether similar events are induced during the estrous cycle, after the proestrous prolactin surge. Rats were killed on proestrus or on estrus, and one ovary was frozen for immunohistochemical detection of MCP-1, monocytes/macrophages (ED1-positive), and differentiated macrophages (ED2-positive) and for in situ detection of apoptotic nuclei. Corpora lutea of the current (proestrus) or preceding (estrus) cycle were dissected from the ovaries of additional rats and frozen for the same analyses and for determination of total protein content. In sections of whole ovaries, intensity and distribution of MCP-1 staining were increased in corpora lutea of multiple ages on estrus as compared to proestrus, as were numbers of differentiated macrophages and apoptotic nuclei per high-power field. Sections of isolated corpora lutea showed these increases on estrus, and the number of monocytes/macrophages per high-power field was also significantly increased. Accompanying these inflammatory/immune events, the corpora lutea on estrus showed decreased weight and total protein per corpus luteum, as compared to corpora lutea on proestrus. These changes are consistent with a proposed role for prolactin in the initiation of luteal apoptosis and of a sequence of inflammatory/immune events that accompany regression of the rat corpus luteum during the normal estrous cycle.