Enhanced antitumour activity of 15-residue bovine lactoferricin derivatives containing bulky aromatic amino acids and lipophilic N-terminal modifications.

In a structure-antibacterial activity relationship study of a peptide fragment of bovine lactoferricin consisting of FKCRRWQWRMKKLGA (LFB 17-31), it was revealed that the two Trp residues were important for antibacterial activity. It has further been demonstrated that the size, shape and the aromatic character of the side chains were even more important than the Trp itself. In this study the antitumour effect of a series of LFB 17-31 derivatives are reported, in which the two Trp residues in position 6 and 8 were replaced with the larger non-coded aromatic amino acids Tbt, Tpc, Bip and Dip. The counterproductive Cys in position 3 was also substituted with these larger aromatic residues. In addition, the effect of introducing lipophilic groups of different size and shape in the N-terminal of the LFB 17-31 sequence was addressed. The resulting peptide derivatives were tested for activity against three human tumour cell lines and against normal human umbilical vein endothelial cells and fibroblasts. High antitumour activity by several of the peptides demonstrated that Trp successfully could be substituted by the bulky aromatic residues, and peptides containing the large and rigid Tbt residue in position 6 and/or 8 in LFB 17-31 were the most active candidates. The antitumour effect was even more increased by the Tbt-modified peptides when the three counterproductive amino acids Cys3, Gln7 and Gly14 were replaced by Ala. Enhanced antitumour activity was also obtained by modifying the N-terminal of LFB 17-31 with either long-chained fatty acids or bulky moieties. Thus, our results revealed that the size and shape of the lipophilic groups and their position in the peptide sequence were important for antitumour activity.     

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.     

The dimerization domain of LFB1/HNF1 related transcription factors: a hidden four helix bundle?

LFB1/HNF1 alpha and LFB3/HNF1 beta bind DNA as dimers and form heterodimers together in vivo and in vitro. The dimerization domain has been located in both proteins in the 32 N-terminal residues. In previous papers we have described the conformational stability as determined by CD and the secondary structure by NMR studies of a peptide with the amino acid sequence of the dimerization domain of LFB1/HNF1 alpha. This study presents a more complete characterization of similar synthetic peptides spanning the LFB3/HNF1 beta dimerization domain and the alpha/beta heterodimer. The HNF1 peptides represent an example of structures which cannot be determined by NOE data alone because they are not sufficient to define one unique solution. An approach is presented which combines NMR data, the protein structure database and structural analyses according to known principles of protein structure. On this basis we are able to determine possible solutions and to identify a four helix bundle as the structure most consistent with the experimental evidence.     

Identification of restriction barriers in Pasteurella multocida

Several naturally occurring antibiotic resistance plasmids were isolated from Pasteurella multocida type D strains. One plasmid, pPM1, was used to study transfer of DNA among P. multocida strains, and could be transferred into Escherichia coli and some P. multocida isolates. However, pPM1 could only be transferred into the toxigenic P. multocida LFB3 at very low frequency. Plasmid recovered from the electrotransformants could be transferred to LFB3 at high frequency. These plasmid DNAs were resistant to PstI, and sensitive to DpnI digestion. Sensitivity to DpnI was common to all the P. multocida DNAs, but resistance to PstI was confined to LFB3. Plasmid pPM1 treated with PstI methylase was able to transform LFB3 at an increased frequency compared to unmethylated DNA, suggesting that LFB3 has a restriction system which cleaves at or near PstI sites.     

Comparison of NMR structures and model-membrane interactions of 15-residue antimicrobial peptides derived from bovine lactoferricin.

LFB (FKCRRWQWRMKKLGA-HN2) is a 15-residue linear antimicrobial peptide derived from bovine lactoferricin, which has antimicrobial activity similar to that of the intact 25-residue disulfide-cyclized peptide. Previous alanine-scan studies, in which all of the residues in LFB were individually replaced with Ala, showed that the 2 tryptophan (Trp) residues of LFB were crucial to its antimicrobial activity. When either Trp6 or Trp8 was replaced with Ala (LFBA6 and LFBA8, respectively), these 2 peptides were almost devoid of antimicrobial activity. We determined the structures of LFB, LFBA6, and LFBA8 bound to membrane-mimetic SDS micelles using NMR spectroscopy, and studied their interactions with different phospholipid-model membranes. The membrane interactions of LFB exhibited little correlation with its antimicrobial activity, suggesting that the mechanism of action of LFB involves intracellular targets. However, the much higher antimicrobial activity of LFB compared with LFBA6 and LFBA8 might result, in part, from the formation of energetically favorable cation-pi interactions observed only in LFB. Information about the importance of Arg and Trp cation-pi interactions will provide insight for the future design of potent antimicrobial peptidomimetics.     

Reassignment of the human macrophage colony stimulating factor gene to chromosome 1p13-21.

Macrophage colony stimulating factor (CSF-1) is a member of a family of glycoproteins that are necessary for the normal proliferation and differentiation of myeloid progenitor cells. The human CSF-1 gene has previously been assigned to chromosome 5 using somatic cell hybrids, and further localized to 5q33 by in situ hybridization with a 3H labelled cDNA probe. However, the murine macrophage colony stimulating factor gene (csfm) has been localized to a region on mouse chromosome 3 which was previously shown to be syntenic with the proximal region of 1p and not 5q. Using a human genomic DNA clone that contains the CSF-1 gene, we have localized CSF-1 to chromosome 1p13-21 by fluorescence in situ hybridization. The reassignment of the CSF-1 gene argues against its involvement in myeloid disorders with deletions of the long arm of chromosome 5.     

Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene.

We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprinting, gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation, contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor, which we name NF-E2. NF-E1 is a factor that also binds the promoter and the enhancer (present in the 3' flanking region) of the human beta-globin gene. NF-E2 has not yet been described and although it binds to a sequence containing the Ap1 consensus, it appears to be different from Ap1.     

Assignment of two of the translation initiation factor-4E (EIF4EL1 and EIF4EL2) genes to human chromosomes 4 and 20

The eukaryotic translation initiation factor (eIF-4E) has recently been cloned from human, mouse, and yeast. This polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. We have designed oligonucleotide primers to the 3' untranslated region of the gene encoding eIF-4E and specifically amplified the human gene in human/rodent somatic cell hybrids using the polymerase chain reaction. By this method, one of the human eIF-4E genes (EIF4EL1, eukaryotic translation initiation factor 4E-like 1) has been mapped to human chromosome 4qter-4p15. In addition, we have localized a second eIF-4E gene (EIF4EL2, eukaryotic translation initiation factor 4E-like 2) to human chromosome 20 by Southern blot analysis of mapping panels established from human/rodent somatic cell hybrids.     

Stable limited filamentous bulking through keeping the competition between floc-formers and filaments in balance

Limited filamentous bulking (LFB) was proposed to save aeration energy consumption and enhance the capacity of filaments to degrade substrates with low concentrations in activated sludge systems. Operational parameters favorable for maintaining the LFB state were investigated in an anoxic-oxic reactor treating domestic wastewater. The experiments showed that the LFB state would deteriorate with sharply decreasing temperature, reducing substrate gradients or removing anoxic zones. The balance between filaments and floc-formers could be achieved by controlling dissolved oxygen and sludge loading rates to be in optimal ranges. Eikelboom Type 0041 and CandidatusMicrothrix parvicella were the filamentous bacteria responsible for the LFB state. However, the excess growth of Eikelboom Type 021N and Sphaerotilus natans were observed when serious bulking occurred under low substrate gradients. It was demonstrated that stable maintenance of LFB for energy saving was feasible by process control and optimization.     

Chromosomal mapping of brain-derived neurotrophic factor and neurotrophin-3 genes in man and mouse

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NTF3) are two new members of the nerve growth factor gene family, which play important roles in the development and maintenance of the vertebrate nervous system. Here we describe the assignments of the BDNF and NTF3 gene loci to human and mouse chromosomes and discuss the evolutionary relationship of human chromosomes 11 and 12. BDNF has been mapped to human chromosome 11p15.5-p11.2 and to mouse chromosome 2, and NTF3 to human chromosome 12p and mouse chromosome 6.     

Molecular characterization of a gene of the ''EGF family'' expressed in undifferentiated human NTERA2 teratocarcinoma cells.

A novel human gene, encoding a 188 amino acid polypeptide that contains a region similar to that of the epidermal growth factor, has been isolated. The gene, expressed in undifferentiated human and mouse teratocarcinoma cells, is shut off after inducing the cells to differentiate by treatment with retinoic acid. Introduction of the cDNA under the control of a viral LTR induces transformation of NIH3T3 cells.