Production of tumor necrosis factor/cachectin by human T cell lines and peripheral blood T lymphocytes stimulated by phorbol myristate acetate and anti-CD3 antibody.

The induction of mRNA synthesis and accumulation of TNF/cachectin and lymphotoxin (LT) mRNAs in T leukemic cell lines and freshly isolated T cells were studied by Northern blot analyses. Without stimulation, TNF mRNA was barely detected in four T cell lines (CEM, KE4, MT-1, and SKW-3) and not detectable in Molt-4 and Jurkat cells, while a considerable amount of TNF mRNA was observed in HSB-2 cells. When stimulated by PMA, these T cell lines accumulated varying levels of TNF mRNA. All seven T cell lines expressed LT mRNA when unstimulated and responded well to PMA by increased accumulation of LT mRNA. The calcium ionophore A23187 by itself had no effect on TNF and LT mRNA accumulations in these cell lines. The CD3+ T cell lines did not respond to anti-CD3 mAb T3-II alone. However, A23187 and mAb T3-II further elevated TNF and LT mRNA accumulations in PMA-treated T cell lines. Synergism between PMA and mAb T3-II was modest in the CD3+ cell lines. A slight difference in kinetics of TNF and LT mRNA accumulations was noted. In addition, heterogeneities in TNF and LT expressions by these cell lines in responses to PMA and other stimuli were observed. In monocyte-depleted peripheral blood T cell populations. PMA was able to induce both TNF and LT mRNA syntheses. This effect was potentiated markedly by the addition of anti-CD3 mAb T3-II. This synergistic response to anti-CD3 mAb and PMA provided further evidence that T cells were the source of TNF synthesis in these cultures. There was a difference in the kinetics of TNF mRNA accumulation and that of LT mRNA. Maximal accumulation of TNF mRNA occurred at 4 h while 8-18 h was required for maximal LT mRNA accumulation. IL-2 mRNA accumulated at an intermediate peak time of 4-8 h. Western blot analyses and cytotoxicity assays with L cells as targets indicated that these T cell lines and peripheral blood T cells secreted TNF. These results provide further evidence that human T cells are capable of making TNF as well as LT under appropriate stimulations. Their productions are an integral part of T cell response to activation signals. In addition, it appears that the production of these two closely related molecules is independently regulated.

Control of hsp70 RNA levels in human lymphocytes.

The expression of a hsp70 gene in human cells has previously been shown to be related to the growth state of the cells. As an alternative to in vitro synchronization procedures, we have measured steady-state levels of the RNA for a heat-shock protein 70 (hsp70) in human peripheral blood mononuclear cells (PBMC) that are naturally quiescent in a G0 state. The probe used recognized, on RNA blots, one single band. The levels of this hsp70 RNA are elevated in circulating PBMC and decrease when the cells are incubated with serum, or phytohemagglutinin, or simply when they are incubated in culture medium. The levels of hsp70 RNA decrease within 30 min after in vitro culture, and are accompanied by an increase in the levels of c-fos RNA. These findings, together with other recent reports in the literature, suggest a possible role of the hsp70 proteins in the regulation of cell growth.

Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins.

Synapsins are neuronal phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are believed to function in the regulation of neurotransmitter release. Molecular cloning reveals that the synapsins comprise a family of four homologous proteins whose messenger RNA's are generated by differential splicing of transcripts from two genes. Each synapsin is a mosaic composed of homologous amino-terminal domains common to all synapsins and different combinations of distinct carboxyl-terminal domains. Immunocytochemical studies demonstrate that all four synapsins are widely distributed in nerve terminals, but that their relative amounts vary among different kinds of synapses. The structural diversity and differential distribution of the four synapsins suggest common and different roles of each in the integration of distinct signal transduction pathways that modulate neurotransmitter release in various types of neurons.

Two sites of action for synapsin domain E in regulating neurotransmitter release.

Synapsins, a family of synaptic vesicle proteins, have been shown to regulate neurotransmitter release; the mechanism(s) by which they act are not fully understood. Here we have studied the role of domain E of synapsins in neurotransmitter release at the squid giant synapse. Two squid synapsin isoforms were cloned and found to contain a carboxy (C)-terminal domain homologous to domain E of the vertebrate a-type synapsin isoforms. Presynaptic injection of a peptide fragment of domain E greatly reduced the number of synaptic vesicles in the periphery of the active zone, and increased the rate and extent of synaptic depression, suggesting that domain E is essential for synapsins to regulate a reserve pool of synaptic vesicles. Domain E peptide had no effect on the number of docked synaptic vesicles, yet reversibly inhibited and slowed the kinetics of neurotransmitter release, indicating a second role for synapsins that is more intimately associated with the release process itself. Thus, synapsin domain E is involved in at least two distinct reactions that are crucial for exocytosis in presynaptic terminals.

Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection.

A cDNA copy of the major human heat shock mRNA was cloned. The clone is complementary to the mRNA encoding the major 70-kilodalton heat shock protein as shown by hybrid arrest translation. We utilized the cloned DNA to measure induction of the gene during adenovirus infection. The mRNA increases in abundance approximately 100-fold during a wild-type adenovirus infection but does not increase more than 2-fold during an infection in which there is no E1A gene function [high multiplicity of infection of an E1A (-) mutant]. Furthermore, by measuring transcription in isolated nuclei, we found that the induction was transcriptional and was mediated by the E1A gene product. The induction was not maintained, however. After a peak level was obtained, transcription returned to preinfection levels. This decline was also reflected in the cytoplasmic mRNA abundance indicating a rapid turnover of the heat shock mRNA. This rapid turnover of the heat shock mRNA appears to be induced by the viral infection since the heat shock mRNA was found to be stable when synthesized in an adenovirus-transformed cell line.

Cell cycle control of the human HSP70 gene: implications for the role of a cellular E1A-like function.

The gene encoding the human 70-kilodalton heat shock protein (HSP70) is subject to activation by the adenovirus E1A gene product and appears to be regulated in the absence of heat shock by a cellular activity similar to E1A. Given the relation of E1A to alteration of growth control, we have investigated the expression of the HSP70 gene during the cell cycle. Assay of mRNA levels after release from a thymidine-aphidicolin block revealed a 20-fold increase in mRNA abundance, reaching a peak level in the post-S-phase period. Upon reaching this peak level, the abundance of the mRNA then declined as the cells entered the next cycle. Control of the abundance of the mRNA during the cell cycle appeared to be primarily at the level of transcription as measured in nuclear runoff assays. Very similar results were obtained by analyzing the expression of the HSP70 gene in the adenovirus-transformed 293 cell line. Furthermore, the E1A gene was also found to be cell cycle regulated; the activation and peak level of the E1A mRNA occurred at an earlier time than those of the heat shock mRNA, consistent with, but not proof of, the hypothesis that E1A is responsible for the cell cycle control of the HSP70 expression. We therefore suggest that the E1A-like cellular activity may govern certain aspects of cell cycle transcription.

Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity.

We have employed an antiserum specific to the 70-kilodalton human heat shock protein and a cDNA clone specific to the mRNA for this protein to analyze the expression of the gene under noninducing conditions. Expression of the heat shock gene can be detected in the absence of heat induction, and this uninduced level of expression depends greatly on the particular cell type. For instance, the basal expression of the heat shock gene is at least 50 times higher in HeLa cells than in WI38 cells at both the mRNA and protein levels. We have previously shown that the inducer of transcription of the early adenovirus genes, the E1A gene product, also induces the heat shock gene, suggesting that these genes may be subject to the same regulation. We have, therefore, investigated the control of the adenovirus genes in relation to the cellular control of the heat shock gene. We find that human cells that allow a high level of uninduced expression of the heat shock gene (i.e., HeLa cells) also allow expression of the early adenovirus genes in the absence of the E1A inducer. The same is also true for the mouse F9 teratocarcinoma cell line. F9 stem cells, which constitutively express the heat shock protein, allow early adenovirus gene expression in the absence of E1A; upon differentiation induced by retinoic acid and cyclic AMP, the cells become restrictive and early viral gene expression requires the E1A gene product. Coordinately, upon differentiation there is also a loss of heat shock protein expression.

Selective induction of human heat shock gene transcription by the adenovirus E1A gene products, including the 12S E1A product.

We have previously shown that the human 70-kilodalton heat shock protein gene (hsp70) is induced by the adenovirus E1A gene product and during the S-G2 phase of the cell cycle. In this study, we investigated the effect of E1A on the expression of other human hsp genes. A gene encoding one form of the hsp89 protein (hsp89 alpha) was activated during an adenovirus infection with kinetics similar to those of activation of hsp70. The induction required a functional E1A gene. However, the hsp89 transcript was not cell cycle regulated. Genes encoding another form of hsp89 and the hsp27 protein were not induced by E1A or during the cell cycle. Further examination of hsp70 expression revealed a greater complexity than previously seen. S1 nuclease analysis using an hsp70 cDNA as well as a distinct hsp70 genomic clone demonstrated three related hsp70 transcripts; two were induced by E1A, and one was not. Both of the E1A-inducible genes were regulated during the cell cycle. All three were induced by heat shock. These results suggest common aspects of control among certain members of this family of cellular genes distinct from heat shock control. Finally, using viruses that express the individual E1A proteins, we found that the hsp70 gene is induced by the 12S and the 13S E1A products. The efficiency of induction by the 12S product was somewhat less than that by the 13S product but only by a factor of less than 2. This is in contrast to the induction of early viral genes, for which the 13S product is considerably more efficient than the 12S product.

Synapsin III: developmental expression, subcellular localization, and role in axon formation.

We have investigated the developmental expression and subcellular localization of synapsin III, the newest member of the synapsin family, in cultured mouse hippocampal neurons. Our results indicate that synapsin III is expressed early during development, with levels peaking 7 d after plating and declining thereafter. Synapsin III is highly concentrated in growth cones. Using specific antisense oligonucleotides, we have also examined the effect of depleting synapsin III on neurite elongation and synaptogenesis. When synapsin III was suppressed immediately after plating, hippocampal neurons extended minor processes but failed to differentiate one of them as the axon. The suppression of synapsin III after axonal elongation did not affect the time course of synapse formation. The results indicate that synapsin III has a developmental time course, a subcellular localization, and a developmental function very different from those of synapsin I and synapsin II.

Site-directed mutagenesis of a single residue changes the binding properties of the serotonin 5-HT2 receptor from a human to a rat pharmacology.

Mesulergine displays approximately 50-fold higher affinity for the rat 5-HT2 receptor than for the human receptor. Comparison of the deduced amino acid sequences of cDNA clones encoding the human and rat 5-HT2 receptors reveals only 3 amino acid differences in their transmembrane domains. Only one of these differences (Ser----Ala at position 242 of TM5) is near to regions implicated in ligand binding by G protein-coupled receptors. We investigated the effect of mutating Ser242 of the human 5-HT2 receptor to an Ala residue as is found in the rat clone. Both [3H]mesulergine binding and mesulergine competition of [3H]ketanserin binding showed high affinity for rat membranes and the mutant human clone but low affinity for the native human clone, in agreement with previous studies of human postmortem tissue. These studies suggest that a single naturally occurring amino acid change between the human and the rat 5-HT2 receptors makes a major contribution to their pharmacological differences.

The molecular biology of serotonin receptors. An overview.

Recently, the family of G protein-coupled serotonin (5-hydroxytryptamine[5-HT]) receptors has begun to yield to molecular analysis. The cloning of the 5-HT1C and 5-HT2 receptors has provided a structural basis for the similarities observed in their pharmacologic properties. Furthermore, pharmacologic characterization of the transfected human 5-HT2 receptor has answered two outstanding questions regarding this receptor. First, the few amino acid differences that exist between the human and the rat genes are sufficient to account for the species differences seen in their pharmacologic properties. Second, the single protein encoded by the human 5-HT2 receptor gene is capable of binding both [3H]DOB and [3H]ketanserin. Analysis of the effects of guanine nucleotides provides further evidence that this single protein binds both ligands, that this receptor has high- and low-affinity states, and that these states are partially interconvertible. Furthermore, the close relationship between the adrenergic receptors and the 5-HT1A receptor has been reaffirmed by the recent cloning of a new adrenergic receptor subtype, alpha 2B, by use of the 5-HT1A receptor sequence. Finally, the detailed level of structural information now available on serotonin receptors has yielded valuable information about the ligand binding site and about the possible functional significance of differing rates of evolutionary change in various parts of the gene.

Brain specific proteins binding to the 3' UTR of the 5-HT2C receptor mRNA.

The 5-HT2C receptor2 is a prominent serotonin receptor that is uniquely expressed in the central nervous system and has been implicated in a variety of psychiatric diseases. While characterizing the 5-HT2C receptor gene, we observed that the mRNA contains a long 3' untranslated region that binds multiple brain proteins. Two proteins, molecular weights 55 and 58 kDa, were of particular interest because they were detected only in brain regions known to express the 5-HT2C receptor abundantly, namely, the hippocampus and cortex. These proteins bind with high affinity to the 5-HT2C receptor mRNA at its extreme 3' end (Kd = 1.8 nM), and binding can be specifically competed by selected regions of the 3' UTR. Furthermore, binding of the 55 and 58 kDa proteins to the mRNA is directionally specific and shows preference for an AU-rich loop containing 6 to 7 nucleotides. These results suggest the possibility that these two brain specific proteins may play a role in the post-transcriptional regulation of the 5-HT2C receptor, and that post-transcriptional control of 5-HT2C receptor expression may be an important regulatory mechanism which has not been previously reported for this serotonin receptor subtype.

Influenza A virus infection in infants.

Influenza A virus causes a variety of respiratory and nonrespiratory illness in children. The symptomatology varies with different age groups. The purpose of this retrospective study was to define the clinical characteristics of influenza A infection in Taiwanese infants. During the period from December 1997 to February 1998, 37 febrile patients younger than 1 year of age, including five newborns, were admitted to our hospital due to suspicion of sepsis or meningitis. The medical records of these patients were retrospectively evaluated. Influenza A virus was isolated from the specimens of the throat swabs in all patients, whereas no bacterial pathogen was detected. The most common clinical manifestations of these infants were lower respiratory tract infections, including pneumonia, bronchiolitis, and croup. There was no significant difference between the clinical characteristics of infants younger than 3 months and those aged from 3 months to 1 year. The mean duration of fever, peak of body temperature, and duration of hospitalization were 3.41 (+/-1.86) versus 4.4 (+/-2.02) days, 39.0 (+/-0.57) versus 39.9 (+/-0.63) oC, 4.9(+/-1.49) versus 6.3 (+/-3.7) days in infants younger than 3 months and infants aged from 3 months to 1 year, respectively. The older infants aged from 3 months to 1 year had a significantly higher peak body temperature than the infants younger than 3 months (p < 0.05). Two patients with croup had a more severe clinical course, however, the outcomes were good in all patients. During an influenza A virus outbreak, influenza A infection should be included in the differential diagnosis of infants with lower respiratory tract infection.

Kawasaki disease presenting as cervical lymphadenitis or deep neck infection.

OBJECTIVE: To describe a group of patients with Kawasaki disease who had cervical lymphadenopathy as their dominant initial presentations. MATERIALS AND METHODS: We retrospectively reviewed the medical records of 14 children who were admitted to Chang-Gung Children's Hospital between May 1996 and July 1998 with the initial impression of cervical lymphadenitis, cellulitis, and/or deep neck infection but for which a diagnosis of Kawasaki disease was established later. RESULTS: Five (35.7%) patients were less than 5 months of age, and 8 (57.1%) patients were more than 53 months of age. The mean duration for establishing a diagnosis of Kawasaki disease from the onset of illness was 8.2 (6 to 20) days. Initially, empiric antibiotics were prescribed in each case with unsatisfactory response. Intravenous immune gamma globulin (2 g/kg) was administered in 13 patients. Three (21.4%) patients developed coronary artery lesions. CONCLUSION: If a child less than 6 months or more than 4 years of age has a fever and an enlarged cervical lymph node and is unresponsive to empiric antibiotics, Kawasaki disease should be considered.

Cloning of cDNAs encoding human synapsins IIa and IIb.

The synapsins are a family of neuronal phosphoproteins that are specifically associated with the cytoplasmic surface of synaptic vesicles. In mammals, distinct genes for synapsins I, II, and III give rise to members of the synapsin family. The synapsins are implicated in neurotransmitter release and synaptogenesis, processes believed to be aberrant in several neuropsychiatric diseases. The characterization of human synapsins is therefore important for evaluating the possible role of synapsins in human neuropathology. In this report, we describe the cloning and sequence of human synapsins IIa and IIb, products of the synapsin II gene. Human synapsins IIa and IIb conform to the previously described domain model of the synapsins, and the most conserved protein domains are A, C, and E.

Isoforms of the neuronal glutamate transporter gene, SLC1A1/EAAC1, negatively modulate glutamate uptake: relevance to obsessive-compulsive disorder.

The SLC1A1 gene, which encodes the neuronal glutamate transporter, EAAC1, has consistently been implicated in obsessive-compulsive disorder (OCD) in genetic studies. Moreover, neuroimaging, biochemical and clinical studies support a role for glutamatergic dysfunction in OCD. Although SLC1A1 is an excellent candidate gene for OCD, little is known about its regulation at the genomic level. Here, we report the identification and characterization of three alternative SLC1A1/EAAC1 mRNAs: a transcript derived from an internal promoter, termed P2 to distinguish it from the transcript generated by the primary promoter (P1), and two alternatively spliced mRNAs: ex2skip, which is missing exon 2, and ex11skip, which is missing exon 11. All isoforms inhibit glutamate uptake from the full-length EAAC1 transporter. Ex2skip and ex11skip also display partial colocalization and interact with the full-length EAAC1 protein. The three isoforms are evolutionarily conserved between human and mouse, and are expressed in brain, kidney and lymphocytes under nonpathological conditions, suggesting that the isoforms are physiological regulators of EAAC1. Moreover, under specific conditions, all SLC1A1 transcripts were differentially expressed in lymphocytes derived from subjects with OCD compared with controls. These initial results reveal the complexity of SLC1A1 regulation and the potential clinical utility of profiling glutamatergic gene expression in OCD and other psychiatric disorders.

Mice lacking synapsin III show abnormalities in explicit memory and conditioned fear.

Synapsin III is a neuron-specific phosphoprotein that plays an important role in synaptic transmission and neural development. While synapsin III is abundant in embryonic brain, expression of the protein in adults is reduced and limited primarily to the hippocampus, olfactory bulb and cerebral cortex. Given the specificity of synapsin III to these brain areas and because it plays a role in neurogenesis in the dentate gyrus, we investigated whether it may affect learning and memory processes in mice. To address this point, synapsin III knockout mice were examined in a general behavioral screen, several tests to assess learning and memory function, and conditioned fear. Mutant animals displayed no anomalies in sensory and motor function or in anxiety- and depressive-like behaviors. Although mutants showed minor alterations in the Morris water maze, they were deficient in object recognition 24 h and 10 days after training and in social transmission of food preference at 20 min and 24 h. In addition, mutants displayed abnormal responses in contextual and cued fear conditioning when tested 1 or 24 h after conditioning. The synapsin III knockout mice also showed aberrant responses in fear-potentiated startle. As synapsin III protein is decreased in schizophrenic brain and because the mutant mice do not harbor obvious anatomical deficits or neurological disorders, these mutants may represent a unique neurodevelopmental model for dissecting the molecular pathways that are related to certain aspects of schizophrenia and related disorders.