Neuropeptide gene expression and neural activity: Assessing a working hypothesis in nucleus caudalis and dorsal horn neurons expressing preproenkephalin and preprodynorphin

1. The working hypothesis that neuropeptide gene expression in a neuron is an indicator of that neuron's physiological activity is discussed. 2. Representative examples from the literature are presented to support the hypothesis. 3. Further, we discuss the regulation of expression of two opioid peptides, preproenkephalin and preprodynorphin, in laminae I and II of the spinal cord and in nucleus caudalis of the trigeminal nuclear complex, where they may play a role in pain modulation. 4. The expression of the opioid peptide genes can be induced by both painful and nonnoxious stimuli in neurons in time-dependent and sensory-specific fashions.     

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.Abbreviations AcNPV Autographica californica nuclear polyhedrosis virus - HSV Herpes Simplex Virus - IE Immediate Early - moi multiplicity of infection - Sf9 Spodoptera frugiperda cells     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes

Melanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.     

Dioecism of the genusCallicarpa (Verbenaceae) in the Bonin (Ogasawara) Islands

The morphology and function of the flowers of three species of the genusCallicarpa, C. glabra, C. nisnimurae andC. subpubescens, endemic to the Bonin Islands, revealed that all three species were dioecious. Male plants had short-styled flowers with sterile ovaries, while female plants had long-styled flowers with non-germinating inaperturate pollen grains. Such dioecism has never been reported from the family Verbenaceae. This curious sexual system with non-germinating pollen grains may have been maintained as a reward to insect pollinators.     

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human -interferon (-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.Abbreviation HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Transfilter analysis of the inductive influence of proventricular mesenchyme on stomach epithelial differentiation of chick embryos

Summary To clarify the precise conditions under which chick embryonic proventricular mesenchyme can induce proventricular epithelial differentiation, transfilter experiments were carried out. Six-day proventricular epithelium formed glands and expressed pepsinogen when a Nucleopore filter with a pore size of more than 0.6 m, but not 0.2 m, was inserted between the epithelium and the proventricular mesenchyme. The larger the pore size of the filter, the more elongated the glands and the more pepsinogen was induced in the explants. The quail nuclear marker and scanning electron microscopy were used to examine penetration of mesenchymal cells through the Nuclepore filter. The filter of more than 0.2 m pore size allowed cell processes of mesenchymal cells to pass through. However, only the filter with a pore size of more than 0.6 m allowed actual migration of mesenchymal cells through the filter, and the larger the pore size of the filter, the more mesenchymal cells passed through. Under the same conditions 6-day and 4.5-day gizzard epithelium formed glands and expressed pepsinogen. These results indicate that a flow of diffusible substances through a Nuclepore filter and even direct contact of a few short cell processes of mesenchymal cells with epithelial cells are not sufficient for induction, and that direct contact of mesenchymal cell processes and/or mesenchymal cells with epithelial cells over a considerably wide area may be prerequisite for the induction.