Two peptides derived from the nerve growth factor precursor are biologically active.

This report provides evidence that the proregion of the NGF precursor protein contains two novel bioactive peptides. The presence of pairs of basic amino acid (aa) residues in the NGF proregion suggests that two or three peptides other than NGF may be generated by proteolytic cleavage. Synthetic peptides of 29 aa (LIP1) and 38aa (LIP2) corresponding to the sequences -71 to -43 and -40 to -3 of the proNGF, respectively, were used in this study. ELISA specific for these two peptides revealed their presence in the rat intestine. LIP1 was localized by immunohistochemistry in endocrine cells of the intestinal epithelium, and LIP2 was immunoprecipitated from an intestinal extract. We also provide evidence for the presence of specific receptors for LIP2 in several cell lines. Scatchard analysis indicated the presence of a low affinity binding site with a Kd of approximately 10(-7) M and a high affinity binding site of 10(-9) M. Cross-linking studies revealed receptor forms of about 140 kD and 93 kD in a prostatic adenocarcinoma cell line. LIP1 and LIP2 induced rapid F-actin redistribution in PC12 cells within 2 min of incubation, which suggests a role of LIP1 and LIP2 in the process of neurite outgrowth. Furthermore, both propeptides induced rapid tyrosine phosphorylation of the Trk protein in both prostatic adenocarcinoma cells and PC12 cells, thus implicating trk in their mechanism of action. These results support our hypothesis that two peptides within the NGF precursor protein are biologically active.

Inhibition of lysophosphatidic acid-induced neurite retraction and cell rounding by SR 57746A.

Rapid neurite retraction and transient rounding of serum-starved NG108-15 and PC12 cells by lysophosphatidic acid (LPA) is retarded and reduced by pre-incubation of the cells with the small non-peptidic molecule, SR 57746A, which exhibits neurotrophic properties. The compound also antagonizes the redistribution of filamentous actin by LPA in both cell types. We hypothesize that the SR 57746A attenuation of LPA-induced effects may account for at least some of the neuroprotective properties of this molecule.

Interleukin-13 is a monocyte chemoattractant.

Recombinant human IL-13 is chemotactic for purified human peripheral blood monocytes Cell migration is stimulated with a typical bell-shaped concentration curve and is maximal at 10 ng/ml. Migration is the result of chemotaxis, and not chemokinesis, as shown by checkerboard experiments. The chemotactic activity of IL-13 on monocytes is not inhibited by preincubation of the cells with pertussis toxin but is diminished by preincubation with protein kinase inhibitors. The related cytokine, IL-4, also stimulates migration of monocytes in the Boyden chamber assays at concentrations similar to those effective for IL-13. Human IL-13 is capable of attracting rabbit peripheral blood monocytes at those concentrations active on human monocytes. On the other hand, no neutrophil migration was induced by IL-13, even at 1 microM concentrations.

The biological activities of gro beta and IL-8 on human neutrophils are overlapping but not identical.

Gro beta and IL-8 are two members of the small induced secreted (SIS) cytokine family (C-X-C subgroup) with proinflammatory activities on neutrophils. In order to assess whether or not the interaction with their receptors results in similar biological actions, we compared the two cytokines in five different bioassays. Gro beta showed similar biological activities as IL-8 in tests of chemotaxis, induction of the respiratory burst, and induction of interleukin 6 (IL-6) production. However, for two other biological activities: augmentation of the expression of CD11b on the cell surface and rapid elevation of the intracellular calcium concentration, maximal effects required 100 times more gro beta than IL-8. Taken together, these results suggest that the stimulation of the IL-8 or gro beta receptor evokes three similar responses, but that only the activation of the IL-8 receptor and not that of gro beta results in elevated CD11b expression and calcium mobilization in human neutrophils.

Functional linkage of the Gro beta and IL-8 receptors on the surface of human neutrophils.

Gro beta and IL-8 are two pro-inflammatory cytokines with chemotactic activities on neutrophils. Binding studies were performed to ascertain whether their similar biological activities are mediated through the same receptor. Since Gro beta lacks tyrosine residues, recombinant Gro beta containing an additional carboxyterminal tyrosine residue (Gro beta-Tyr) was produced in transfected COS cells, purified to homogeneity and radiolabelled with 125INa. Saturation experiments using [125I]-Gro beta-Tyr allowed us to identify high affinity receptors on human neutrophils (Kd: 2 +/- 0.5 nM and Bmax: 4760 +/- 761 sites/cell). Experiments using [125I]-IL-8 as ligand, showed no significative differences in affinity (Kd: 4 +/- 0.9 nM) but about two times the number of sites (11316 +/- 1810 sites/cell). In competition experiments using [125I]-Gro beta-Tyr, unlabelled IL-8 and Gro beta-Tyr generated superposable displacement curves (IC50: 0.69 +/- 0.15 nM and 0.42 +/- 0.11 nM, respectively). Interesting, IL-8 binding sites could be down-regulated by Gro beta and IL-8, indicating that the two binding sites may be associated. Cross-linking experiments using [125I]-IL-8 revealed two major bands at 70 and 140 kDa, whereas experiments with [125I]-Gro beta-Tyr showed only the 70 kDa band. Taken together, these results suggest that the human neutrophil IL-8/Gro beta receptor is a dimeric complex with two high affinity binding sites for IL-8 and of those two, only one is shared by Gro beta.

Molecular cloning of the MCP-3 chemokine gene and regulation of its expression.

We have isolated a cDNA (NC28) transcribed from a mRNA which is transiently induced in U937 promonocytic cells by PMA and super-induced by cycloheximide. NC28 cDNA encodes a new member of the chemokine family, MCP-3, recently purified from MG-63 osteosarcoma cells by Van Damme et al. [1]. The MCP-3 protein sequence shows 74% identity with human monocyte chemoattractant protein 1 (MCP-1) and, like MCP-1, recombinant MCP-3 protein shows chemotactic activity for monocytes but not for neutrophils. However the secreted MCP-3 protein differs from MCP-1 in being N-glycosylated. The 3' noncoding regions of MCP-3 and MCP-1 mRNAs are more diverged (44%), allowing specific cDNA probes to be made, and indicating that the two genes are evolutionarily distant. Sequence comparisons of the 3' noncoding regions suggest that MCP-3 may be the human homologue of the mouse MARC gene [2], and that MCP-1 and MCP-3 genes arose by a gene duplication event before the mammalian radiation. Both MCP-1 and MCP-3 mRNAs are expressed by PBMC, principally by monocytes, with MCP-1 mRNA being expressed at levels 2-4 times that of MCP-3 mRNA. However, while MCP-1 mRNA is also expressed at high levels in fibroblast or astrocytoma cell lines after IL-1 and TNF stimulation, MCP-3 mRNA is expressed only at very low levels in these cells. The cellular origin of MCP-3 is thus more restricted than that of MCP-1. In our experiments on PBMC, LPS is not a consistent inducer of MCP-1 and MCP-3 mRNAs. In some experiments, it actually decreases levels of these two mRNAs, while concomitantly increasing IL-6 and TNF-alpha mRNA levels. Levels of MCP-1 and MCP-3 mRNAs in PBMC are both increased by IFN-gamma, although IL-6 mRNA is not induced. They are also increased by PHA-P and are decreased, in most cases, by IL-13 [3]. MCP-1 and MCP-3 mRNAs are thus co-ordinately regulated in monocytes in response to a number of inducing or inhibitory agents, in a manner differing in several respects from that of other monokines such as IL-6.

Cloning and biosynthetic studies of rat somatostatin.

The predicted amino acid sequence of rat preprosomatostatin has been obtained by cloning and subsequent DNA sequence analysis of a cDNA obtained from mRNA prepared from a rat medullary thyroid carcinoma (MTC). The predicted preprosomatostatin is 116 amino acid residues in length. Somatostatin-14 is located at the C-terminus of the preprohormone and the amino terminus contains a 'signal' peptide of 24 amino acids. A somatostatin amino terminal protein of 78 residues is found between the signal peptide and somatostatin-14. Both somatostatin-14 and somatostatin-28 are observed in the thyroid tumour C-cells. A comparison of the rat and human preprosomatostatin amino acid sequences shows only 4 substitutions observed in 116 amino acids. Patients with localized MTC often exhibit high serum calcitonin levels while patients showing cellular heterogeneity in the MTC appear to have lower calcitonin levels and a virulent neoplasia with a grave prognosis. Numerous rat MTCs have been examined by two dimensional gel electrophoresis. It is possible to distinguish characteristic differences in protein profiles of tumours producing high levels of calcitonin from those showing low calcitonin and high somatostatin levels. This analysis can be done with less than 1 mg of tissue and may represent a valuable prognostic tool in evaluating the clinical variability of MTC.

Bacteriophage T7 DNA replication: a linear replicating intermediate (gradient centrifugation-electron microscopy-E. coli-DNA partial denaturation).

The T7 chromosome in the first round of replication is a Y-shaped DNA rod. Thus, it differs from previously observed bacterial and viral replicating chromosomes that are circular.

Initiation and reinitiation of DNA synthesis during replication of bacteriophage T7.

In its first round of replication, the T7 chromosome follows a simple pattern, as viewed in the electron microscope. The iniation of DNA synthesis occurs about 17% from the genetic left end of the viral DNA rod. Bidirectional DNA synthesis from this origin then generates a replicating intermediate that we call an "eye form." In the eye form, when synthesis in the leftward direction reaches the left end of the viral chromosome, the molecule is converted into a Y-shaped replicating rod. The remaining growing point continues synthesis rightward, until presumably it runs off the right end of the DNA rod, thus terminating replication. Numerous T7 chromosomes were found in which a second round of replication had begun before the first round had finished. Analysis of these reinitiated DNA molecules showed that the second round of replication, like the first, began 17% from the end of the chromosome and involved bidirectional DNA synthesis.

Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain.

Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.

Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain.

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin.

Strand-specific viral DNA synthesis in purified viroplasms isolated from turnip leaves infected with cauliflower mosaic virus.

There is some evidence that two steps are involved in the DNA replication of cauliflower mosaic virus (CaMV): the first one may occur in the nucleus and the second one in the cytoplasm of infected cells. The latter would correspond to the reverse transcription step recently proposed in the model of the viral life cycle, and could occur in the viroplasms which are CaMV-induced cytoplasmic inclusion bodies. In order to test whether viroplasms are capable of DNA synthesis and to characterize the associated enzymatic activities, we developed an extensive purification method for these organelles. Such isolated viroplasms are indeed able to incorporate radioactive precursors into exclusively viral-specific sequences without added template primer. Hybridization of sequences labeled in viroplasms to cloned CaMV DNA shows that the DNA synthesis occurs throughout the whole viral genome and has marked strand specificity; neosynthesized molecules are of minus polarity, i.e., complementary to the large viral transcript (35 S RNA). Moreover, during the purification of viroplasms, the poly(rC)-directed DNA synthesis activity, which is specific to infected plants, is preferentially retained.

Novel bacteriophage lambda mutation affecting lambda head assembly.

A novel phage lambda mutation, called dc10, which interferes with proper lambda head assembly has been isolated and characterized. Phage lambda carrying this mutation is (i) unable to form plaques at 30 or 37 degrees C but does so at 42 degrees C and (ii) unable to form plaques at 42 degrees C on pN-constitutive hosts. Both properties are due to dc10 since all phage revertants for one phenotype simultaneously lose the other phenotype and vice versa. The dc10 mutation has been mapped in the B gene and has been shown to be dominant over the corresponding wild-type product. At 30 degrees C the dc10 mutation results in the formation of abnormal petit lambda heads made up of pE, pB, pC, and pNu3. Under pN-constitutive conditions, the dc10 mutation results in the formation of abnormal petit lambda heads made of pE, X1, and X2 only. A model to explain the data is presented.

Evidence for nerve growth factor-potentiating activities of the nonpeptidic compound SR 57746A in PC12 cells.

SR 57746A (1-[2-(naphth-2-yl)ethyl]-4-(3-trifluoromethylphenyl)-1,2,5,6- tetrahydropyridine hydrochloride) exhibits neurotrophic activities in vivo and in vitro. We used the rat pheochromocytoma PC12 cell line to investigate in vitro cellular changes induced by SR 57746A. A significant increase in the percentage of cells bearing neurite-like processes was obtained in cells treated by SR 57746A and nerve growth factor (NGF) compared with NGF treatment alone. SR 57746A added alone, however, had no effect on morphogenesis or on survival of cells in serum-free medium. In contrast, SR 57746A induced a "priming" effect on PC12 cells for neurite outgrowth within 6 h of addition of the protein tyrosine kinase inhibitor genistein. An increase in alpha-actinin content resulted from treatment with SR 57746A. Expression of NGF-mediated acetylcholinesterase and choline acetyltransferase was enhanced within 5 days by SR 57746A. The molecule also induced rapid F-actin redistribution. Within 2 min of incubation, outgrowth of F-actin-containing filopodia was clearly visible at the cell periphery, as previously shown with NGF. It is interesting that this effect of SR 57746A could be mimicked by protein tyrosine kinase inhibitors and abolished by preincubation with sodium orthovanadate, a protein tyrosine phosphatase inhibitor.

Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples.

Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2.

We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and the structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2.

Sequence of a cDNA encoding pancreatic preprosomatostatin-22.

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr, 11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length of poly(A). The putative preprohormone contains a sequence of hydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence of approximately 57 amino acids is followed by a single Arg-Arg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection of mRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on Na-DodSO4/polyacrylamide gels.

Isolation, characterization, and DNA sequence of the rat somatostatin gene.

The gene encoding rat somatostatin has been isolated from a lambda phage gene library. Phage harboring the gene were identified by plaque hybridization using a nick-translated fragment derived from the cDNA for rat somatostatin. The transcriptional unit includes exons of 238 and 367 base pairs (bp) separated by one intron of 621 bp. The intron is located between the codons for Gln (-57) and Glu (-56) of prosomatostatin. Analysis of the nucleotide sequence 5' to the start of transcription reveals a number of sequences which may be involved in the expression of somatostatin. A variant of the "TATA" box, TTTAAA, lies 26 bp upstream from the start of transcription, and a sequence homologous to the "CAAT" box (GGCTAAT) is 92 bp upstream from the transcription start. A long alternating purine-pyrimidine stretch, (GT)25, which is similar to Z DNA-forming sequences in other genes, lies 628 bp 5' to the transcription start and is flanked by small repeats. Hybridization analysis shows that this region is highly repeated in the genome and that homologous sequences are located approximately 2 kilobase pairs downstream from the poly(A) addition site. Southern hybridization of the lambda clone with probes derived from brain or liver poly(A+) RNA demonstrates that another transcribed sequence lies about 7 kilobase pairs downstream from the poly(A) addition site of the rat somatostatin gene. Analysis of rat DNA suggests that there may be restriction-site polymorphisms in or near the gene or that additional somatostatin-hybridizing sequences may exist in the genome.

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals.

Receptors and transduction pathways for monocyte chemotactic protein-2 and monocyte chemotactic protein-3. Similarities and differences with MCP-1.

MCP-2 and MCP-3 are recently identified members of the Cys-Cys chemokine family with high sequence similarity with MCP-1 (62% and 71%, respectively). The present study was aimed at defining receptor usage and signal transduction pathways of MCP-2 and MCP-3 in human monocytes in comparison with MCP-1. MCP-2 and MCP-3 induced migration of monocytes with a typical bell-shaped curve and maximal response at 10 and 50 ng/ml, respectively. The maximal response elicited by MCP-2 and MCP-3 was lower (approximately 60%) than that of MCP-1. Pertussis toxin (PTox) inhibited the chemotactic activity of MCP-3 and MCP-1 (IC50 = 6.2 and 4.4 ng/ml, respectively), whereas cholera toxin (CTox) had little effect on these two chemokines (IC50 > 1000 ng/ml). In contrast, MCP-2-induced chemotaxis was blocked by CTox (IC50 = 75 ng/ml) and relatively unaffected by PTox. MCP-3 and MCP-1 induced a rapid increase in intracellular Ca2+ concentration, whereas MCP-2, in the range of concentrations active on chemotaxis, did not. MCP-1-, MCP-2-, and MCP-3-induced chemotactic responses were blocked by C-I, a serine/threonine kinase inhibitor, and by genistein, a tyrosine kinase inhibitor, with the MCP-2 response being more sensitive than those induced by MCP-1 and MCP-3. MCP-1 and MCP-3 rapidly induced arachidonic acid release whereas MCP-2 was ineffective. MCP-1 and MCP-3 cross-desensitized with each other in terms of Ca2+ transients and displaced with a comparable efficiency labeled MCP-1 from human monocytes. On the other hand, MCP-2 did not cross-desensitize with MCP-1 and MCP-3 and only partially (20%) displaced labeled MCP-1. Thus, in spite of high sequence similarity, MCP-2 differed considerably from MCP-1 and MCP-3 in terms of sensitivity to CTox and PTox, arachidonate and calcium mobilization, and capacity to compete for labeled MCP-1.