Exploration of surfactin production by newly isolated Bacillus and Lysinibacillus strains from food-related sources.

As a lipopeptide (LP), surfactin exhibits properties, such as emulsifying and dispersing ability, which are useful in food industry. Discovery of new LP-producing strains from food sources is an important step towards possible application of surfactin in foods. A total of 211 spore-forming, Gram-positive, and catalase-positive bacterial strains were isolated from fermented African locust beans (iru) and palm oil mill effluents in a screening process and examined for their ability to produce surfactin. This was achieved by a combination of methods, which included microbiological and molecular classification of strains, along with chemical analysis of surfactin production. Altogether, 29 isolates, positive for oil spreading and emulsification assays, were further identified with 16S rDNA analysis. The strains belonged to nine species including less commonly reported strains of Lysinibacillus, Bacillus flexus, B. tequilensis, and B. aryabhattai. The surfactin production was quantitatively and qualitatively analysed by high-performance thin-layer chromatography and liquid chromatography-mass spectrometry (LC-MS). Confirmation of surfactin by MS was achieved in all the 29 strains. Highest surfactin production capability was found in B. subtilis IRB2-A1 with a titre of 1444·1 mg L-1 .

The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea.

Here we show that the mature cochlear neurons are a rich source of acidic fibroblast growth factor (aFGF), which is expressed in the neuronal circuitry consisting of afferent and efferent innervation. The site of action of neuronal aFGF is likely to reside in the organ of Corti, where one of the four known FGF receptor (FGFR) tyrosine kinases--namely, FGFR-3 mRNA--is expressed. Following acoustic overstimulation, known to cause damage to the organ of Corti, a rapid up-regulation of FGFR-3 is evident in this sensory epithelium, at both mRNA and protein levels. The present results provide in vivo evidence for aFGF being a sensory neuron-derived, anterogradely transported factor that may exert trophic effects on a peripheral target tissue. In this sensory system, aFGF, rather than being a neurotrophic factor, seems to promote maintenance of the integrity of the organ of Corti. In addition, aFGF, released from the traumatized nerve endings, may be one of the first signals initiating protective recovery and repair processes following damaging auditory stimuli.

Acidic FGF and FGF receptors are specifically expressed in neurons of developing and adult rat dorsal root ganglia.

Employing complementary technical approaches, we have studied the expression of acidic fibroblast growth factor (aFGF) and FGF receptors in rat dorsal root ganglia. The results clearly showed that within spinal nerves aFGF and two high-affinity FGF receptors, FGFR-1 and FGFR-2, were prominently expressed in neurons, while expression in Schwann cells was undetectable. FGFR-3 and FGFR-4 were not expressed in dorsal root ganglia. Acidic FGF mRNA was detected in the majority of dorsal root ganglion neurons, including all size classes: FGFR-1 and FGFR-2 transcripts were only detected in subpopulations of mainly large and medium size neurons. In subcellular fractionation studies on dorsal root ganglion and spinal root tissue, aFGF was recovered in the soluble fraction and was thus not tightly associated with neuronal membranes. During development FGFR-1 and FGFR-2 mRNAs were found to be present at all stages examined (embryonic days 15-21 and postnatal days 1-120). Acidic FGF mRNA and protein were first detected at embryonic day 18, and their expression then increased progressively up to postnatal levels. In cultures of dorsal root ganglion neurons derived from day 15 embryos, aFGF expression was first detected 3 days after plating. The resulting neuron cultures continued to express aFGF in a Schwann cell-independent manner. In combination, these results indicate that aFGF expression in dorsal root ganglia is initiated and maintained in postmitotic neurons. Furthermore, the data suggest that the physiological function of aFGF in the peripheral nervous system is connected to processes specific to the mature sensory (and motor) system, such as the maintenance and survival of peripheral nerve neurons.

Differential expression of acidic and basic FGF in the rat substantia nigra during development.

Both acidic (aFGF) and basic (bFGF) fibroblast growth factors have been shown to be present in the adult rat ventral mesencephalon and to exert effects on cultured mesencephalic cells. In the present study we have examined the expression of aFGF and bFGF in the rat ventral mesencephalon at various stages of development. bFGF was present at all ages examined [embryonic day 16 (E16) to postnatal day 90 (P90)]. In contrast, aFGF was not detectable at embryonic and early postnatal ages, but was observed at later (P20, P60, P90) postnatal stages. These data suggest that aFGF and bFGF may have functions in mesencephalic dopamine neurones in different stages of development.

Expression of acidic and basic fibroblast growth factors in the substantia nigra of rat, monkey, and human.

The distribution of acidic (aFGF) and basic (bFGF) fibroblast growth factor mRNA and protein were examined in mesencephalon by immunohistochemistry, immunoblot analysis, in situ hybridization histochemistry, and RNA analysis. Coexistence of aFGF or bFGF with tyrosine hydroxylase protein in nigral cells was observed with immunohistochemistry. Both aFGF and bFGF mRNAs were found in the substantia nigra. Unilateral 6-hydroxydopamine lesions of nigrostriatal neurons resulted in a loss of aFGF and tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] mRNA-positive neurons on the lesioned side. The distribution of aFGF mRNA in monkey brain was similar to that seen in the rat. RNA and immunoblot analysis confirmed the presence of both aFGF and bFGF mRNAs and proteins in the substantia nigra of rat, monkey, and human.

Basic FGF is present in dopaminergic neurons of the ventral midbrain of the rat.

Immunocytochemistry procedures and 6-hydroxy-dopamine-induced degeneration of dopamine nerve cells provided evidence that practically all tyrosine hydroxylase immunoreactive (IR) neurons of the substantia nigra and ventral tegmental area contain cytoplasmic basic fibroblast growth factor immunoreactivity (bFGF IR), while many astroglial cells in the neostriatum and substantia nigra contain nuclear bFGF IR. RNA blot analysis demonstrated strong expression of a major 3.7 kb bFGF mRNA in the substantia nigra and ventral tegmental area. These results indicate that bFGF may be a significant growth factor in the DA neurons.

Gene transfer into brain tumor cell lines: reporter gene expression using various cellular and viral promoters.

In the present study we determined the optimal conditions for transferring DNA into rat and human brain tumor cell lines of glial and neuronal origin using electroporation as the transfection method. Gene transfer efficiency was measured in terms of transient chloramphenicol acetyltransferase (CAT) activity and stable neomycin expression. Moreover, the activity of a variety of cellular and viral promoters in brain tumor cell lines of distinct origin was characterized. The results revealed various expression patterns, including glial as well as neuronal specific promoter activity.

The expression of the Autographa californica nuclear polyhedrosis virus genome in insect cells.

This report presents a synopsis of recently published work in our laboratory on the molecular biology of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The following studies have been summarized. (1) On the mode of transcription of the AcNPV genome in insect cells. (2) Translation of proteins encoded in the 81.2 to 85.0 map unit segment of AcNPV. (3) Inserts of insect cell DNA in the AcNPV genome. (4) Expression of influenza (fowl plague) virus haemagglutinin in Spodoptera frugiperda insect cells, and successful immunization of chickens. (5) Synthesis of the influenza virus haemagglutinin in insect larvae by recombinant AcNPV. This insect virus system will continue to serve as a model for research on the molecular biology of insects. Moreover, the baculovirus system has been recognized as a very efficient and safe eukaryotic expression vector.

An insertion of insect cell DNA in the 81-map-unit segment of Autographa californica nuclear polyhedrosis virus DNA.

In this report, a transposonlike insertion of Spodoptera frugiperda insect cell DNA was analyzed in single-plaque isolate E of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The 634-base-pair insertion is characterized by an 18-base-pair terminal inverted repeat and carries an EcoRI site. This additional EcoRI site in the 81-map-unit segment of the DNA of plaque isolate E of AcNPV explains the difference between the EcoRI restriction map of the DNA from this isolate and those of the virus stocks used in other laboratories. Except for this insertion, the nucleotide sequence at the site of insertion in the DNA of plaque isolate E is identical to that of AcNPV E2 (G. E. Smith and M. D. Summers, Virology 89:517-527, 1978). The cellular DNA insertion in the AcNPV genome is represented many times in the S. frugiperda cell genome but has no detectable homology with DNAs from species other than lepidopteran insects. In S. frugiperda cells, the transposonlike insertion sequences are transcribed into cytoplasmic RNA. The transcription of these sequences is initiated within the cellular insertion element. As reported previously (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 61:3048-3057, 1987), in S. frugiperda cells infected with plaque isolate E of AcNPV, at least nine different size classes of AcNPV-specific RNAs are synthesized; in AcNPV E2-infected cells, similar size classes have been detected. The cellular insertion of plaque isolate E provides the initiation site for the synthesis of an additional RNA size class which is transcribed off viral DNA.

Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector.

The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%-2% of the newly synthesized protein.

Overlapping sets of viral RNAs reflect the array of polypeptides in the EcoRI J and N fragments (map positions 81.2 to 85.0) of the Autographa californica nuclear polyhedrosis virus genome.

In several parts of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome, nested sets of overlapping RNAs with common 3' or 5' termini have been recognized. In the present report, the pattern of viral transcription and the arrangement of viral gene products in the region of 81.2 to 85.0 map units were investigated. In this segment of the AcNPV genome, at least nine size classes of viral RNA were identified which ranged in size from 1.3 kilobases (kb) to 4.6 kb and exhibited common 3' termini. The detailed restriction map and the nucleotide sequence of this part of the AcNPV genome were determined. Computer analyses revealed several open reading frames (ORFs) on the rightward-transcribed strand with potential TATA and CAAT signals preceding many of the potential ORFs and the 5' termini of some of the mapped RNAs. The leftward-transcribed strand was devoid of major ORFs. The presumptive polypeptides encoded by the larger ORFs ranged in size from 11.3 to 55.6 kilodaltons (kDa). The amino acid sequence of the presumptive polypeptide encoded by ORF3, a 33.6-kDa molecule, exhibited an unusual, clustered 16-fold repeat of the dipeptide arginine-serine in a protein that showed an overall preponderance of basic amino acids. The results of in vitro translation experiments with hybrid-selected RNAs homologous to internal subfragments of the 81.2- to 85.0-map-unit region yielded polypeptides of approximately 28, 34 to 36, and 48 to 50 kDa, which were close in size to the lengths of the major ORFs derived from the nucleotide sequence. The localizations of individual size classes of RNAs in the 81.2- to 85.0-map-unit region of the viral genome were determined precisely at the 3' and 5' termini by S1 protection analyses. Within a sequence of eight nucleotides, all RNAs had the same 3' terminus, which lay close to multiple polyadenylation signals. The initiation sites of the nine different RNA size classes were precisely mapped. As the cap sites of the smaller RNAs (less than 1.8 kb) were determined by S1 protection analyses, a multitude of RNA initiation sites became apparent. It was also shown that the different RNA size classes in the 81.2- to 85.0-map-unit region were detectable as early as 2 h and at least until 36 to 48 h after infection. In unselected cytoplasmic RNA, the size classes of viral RNAs specific for the EcoRI J fragment were detectable early as well as late after infection, although at early times the larger RNAs were detectable in smaller amounts.(ABSTRACT TRUNCATED AT 400 WORDS)