Proficiency studies on the determination of paralytic shellfish poisoning toxins in shellfish.

Paralytic shellfish poisoning toxins are produced by dinoflagellates. Shellfish filtering these unicellular algae will accumulate the toxins and pose a health risk when consumed by man. In the European Union, paralytic shellfish poisoning toxins in bivalve molluscs are regulated at a maximum content of 80 microg/100 g (91/492/EEC). The current reference method in the European Union is the mouse bioassay, but alternative methods including the liquid chromatography methodology are preferred for ethical reasons. Analyses of suspected shellfish batches revealed, however, unacceptable differences in results reported by a small group of Dutch laboratories all using liquid chromatography methods with precolumn derivatization, followed by fluorescence detection. Therefore, a series of proficiency studies were undertaken among these laboratories. In the first three studies, participants were more or less allowed their own choice of method execution details. This approach yielded unsatisfactory results. A fourth study was then initiated in which a standardized method was mandatory. Two types of test material were used in the fourth study: lyophilized Cardium tuberculatum material containing saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX), and lyophilized mussel material containing dc-STX. The latter material was investigated in an interlaboratory study involving 15 participants and was considered as the reference material. Among the four laboratories, coefficients of variation (ANOVA) for C. tuberculatum material were 10% (n = 11) and 9% (n = 12) for STX and dc-STX, respectively, and for the reference material was 8% (n = 12) for dc-STX. The joint efforts showed that variability in analysis results between laboratories that all apply more or less the same method can be drastically improved if the methodology is rigorously standardized.

Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon).

Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein hormone produced in the anterior pituitary gland. The hormone is essential in the regulation of reproductive processes, such as follicular development and ovulation. It is clinically used for treatment of anovulation and in assisted reproduction technologies such as in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Until recently, the only source for human FSH has been the urine from post-menopausal women. Such a natural source implies limited availability and potential product variability. Thus, we have cloned the genes encoding the alpha- and beta-subunits of human FSH and transfected these into Chinese hamster ovary (CHO) cells. A CHO-clone was isolated capable of secreting intact glycosylated FSH with identical amino acid sequences to natural FSH. This cell line was grown in perfusion culture and enabled us to isolate highly pure FSH (> 99%). The complexity of the charge distribution of human recombinant FSH was demonstrated by Isoelectric focusing. The observed microheterogeneity is caused by the large number of carbohydrate chain structures which are added to the four potential glycosylation sites in the alpha beta-dimer. Furthermore, the carbohydrates show a variation in their degree of sialylation which reflects the different pl values of the individual isohormones. Despite the complexity of post-translational modification, the isoform distribution of recombinant FSH produced in a CHO-cell line and grown in perfusion culture is surprisingly similar to that observed with pituitary FSH and urinary FSH. In conclusion, we have shown that FSH-gene transfected CHO-cells are capable of stable serum-free production of recombinant FSH. A process has been developed which assures the consistent and reproducible production of highly-purified recombinant FSH.

Efficient generation of human anti-cytomegalovirus IgG monoclonal antibodies from preselected antigen-specific B cells.

Human anti-cytomegalovirus (CMV) specific B cells were enriched to a purity of up to 38% on CMV-coated dishes and subsequently clonally expanded in the presence of human T cell supernatant and irradiated murine thymoma helper cells. The expanded cells were immortalized by a mini-electrofusion with K6H6B5 myeloma cells. Twenty-two anti-CMV positive B cell clones could be obtained from as little as 1.5 ml donor blood. The majority of these clones produced anti-CMV antibodies of the IgG class. Ten anti-CMV positive B cell clones were submitted to separate mini-electrofusions yielding stable, human anti-CMV IgG-producing hybridomas in six out of ten fusions. These antibodies recognized different proteins of the CMV virus, as deduced from the immunofluorescence staining pattern on infected human fibroblasts.

Immortalization of antigen selected B cells.

This paper reports the generation of monoclonal antibody producing hybridomas from a small number of antigen-specific B cells selected by panning on antigen-coated dishes and rosetting with antigen-coupled paramagnetic beads. Anti-HIV positive B cells from spleen could be recovered by panning with an efficiency of 5% and a purity of 24%. Immunobead selection of anti-HIV positive B cells from the same mice yielded a recovery of 17% and a purity of 7%. Various experimental conditions with respect to the selection of specific B cells were investigated, leading to an optimized protocol for the isolation of a limited subset of B cells. The selected cells retained their property to produce immunoglobulins and could be clonally expanded in the presence of human T cell supernatant and irradiated murine thymoma helper cells to generate sufficient cells for a mini-electrofusion with NS-1 myeloma cells. Up to 78 specific hybridomas could be generated from one anti-HIV positive B cell. An overall efficiency of specific B cell immortalization of up to 10% was obtained.

Comparative in vitro and in vivo studies on the biological characteristics of recombinant human follicle-stimulating hormone.

The in vitro and in vivo activities of recombinant human FSH (recFSH) produced by a Chinese hamster ovary cell line were studied and compared with those of natural FSH preparations. The specific FSH activities of recFSH established by immunoassay and in vivo bioassay were greater than 10,000 IU/mg protein and considerably higher than the activities of tested urinary FSH references, while the in vivo bio/immuno ratios of these preparations were not significantly different. Compared to a highly purified pituitary standard (IS 83/575), recFSH had a comparable high specific in vivo bioactivity, but the specific immunoreactivity of IS 83/575 was about 2 times lower. In receptor displacement and in vitro bioassay studies recFSH provided dose-response curves parallel to those of pituitary and urinary FSH references. When equal amounts of immunoreactivity FSH were tested, recFSH and urinary and pituitary FSH displayed comparable activities in both assays. The in vitro bioactivity of recFSH could be neutralized effectively by each of three monoclonal antibodies raised against recFSH (alpha-specific), urinary FSH (beta-specific), and pituitary FSH (alpha beta-specific), respectively. Moreover, 50% inhibition of comparable responses induced by recFSH, urinary "pure" FSH, or pituitary FSH was established by the same amount of monoclonal antibody. These results support the structural and functional similarity of recFSH and natural FSH. To test whether recFSH is capable of inducing LH-specific biological responses, the in vitro induction of testosterone production in mouse Leydig cells was assessed. At least 16 IU recFSH/ml incubate were needed to increase testosterone production, indicating that the intrinsic LH bioactivity of recFSH is negligible (less than 0.025 mIU LH/IU FSH). The in vivo efficacy of recFSH was examined by treating immature female hypophysectomized rats during 4 days with recFSH only or with recFSH supplemented with hCG. RecFSH only treatment increased ovarian weight and aromatase activity in a dose-dependent manner. When recFSH dosages providing submaximal responses were supplemented with 1 IU hCG, both ovarian weight and aromatase activity were largely augmented. Neither recFSH nor urinary pure FSH, administered in a high dose was able to increase plasma estradiol levels, while ovarian weight and aromatase activity were increased to the same extent. However, when recFSH was supplemented with only 0.1 IU hCG, a 3-fold increase in median plasma estradiol levels was obtained. These findings support the two-cell two-gonadotropin theory, holding that both FSH and LH are required for estrogen biosynthesis, but also reveal that only very small amounts of LH activity are sufficient to increase estrogen secretion up to measurable plasma levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Study of the genetic organisation of a plant viral RNA genome by in vitro expression of a full-length DNA copy.

The genetic approach for elucidating functions encoded by RNA plant viruses has been hampered by the lack of methods to select desired mutants following random mutagenesis. An alternative might be to copy RNA genomes into DNA and use methods for site-directed mutagenesis to modify specific regions of the DNA copy. Transcription of the DNA copy will subsequently produce viral RNA with desired mutations. We have constructed a full-length DNA copy of the smaller of the two cowpea mosaic virus (CPMV) RNAs, referred to as M RNA. The DNA copy was positioned downstream from the promoter of bacteriophage SP6 and using SP6 RNA polymerase, this copy and two derivatives of it containing a specific deletion and insertion, respectively, have been transcribed into RNA molecules which are efficiently translated in rabbit reticulocyte lysates. The results obtained show that the subsequent in vitro transcription and translation of DNA copies may be a powerful tool to unravel the genetic properties of viral RNA genomes.

Generation of AKR mink cell focus-forming virus: nucleotide sequence of the 3' end of a somatically acquired AKR-MCF.

The 3' end of an AKR-MCF provirus (MCFr35) was cloned and found to be biologically active. Comparison of the nucleotide sequence of MCFr35 with the sequence of other MuLVs revealed that the MCFr35 was most likely derived from the same xenotropic and ecotropic parents, which were involved in the generation of AKR-MCF247. Ecotropic sequences are present around the XbaI site at position 7.9 on the genomic map, and in the long terminal repeat. Most of the T1 oligonucleotide sequences, characteristic for the leukemogenic "class I" MCFs, are also present in MCFr35, with the exception of T1 oligonucleotides 108 and 18. The MCFr35 LTR contains a duplicated enhancer sequence from a xenotropic-like provirus, which is present only once per haploid genome equivalent. The 3' end of MCFr35 consist predominantly of nonecotropic sequences, thereby delimiting the positions of recombination in various MCF viruses.

Generation of AKR mink cell focus-forming viruses: a conserved single-copy xenotrope-like provirus provides recombinant long terminal repeat sequences.

AKV and AKR mink cell focus-forming virus-specific probes from the envelope and long terminal repeat (LTR) regions were prepared for study of the structure of recombinant proviruses in tumor tissues of AKR mice. The results showed that (i) all somatically acquired proviruses possessed, besides a recombinant gp70 gene, an altered U3 LTR; (ii) in a substantial portion of the somatically acquired AKR mink cell focus-forming proviruses, the LTR comprised sequences derived from the same xenotropic-like provirus; (iii) this U3 LTR donating parental provirus (Xeno-dL) was present only once per genome equivalent in several mouse strains; (iv) in the strains containing the Xeno-dL provirus, the provirus was present in the same chromosomal site; (v) restriction analysis of the Xeno-dL revealed that the mink cell focus-forming gp70 sequences were derived from a parental provirus, different from Xeno-dL. Therefore, at least two non-ecotropic parents participate in the generation of leukemogenic AKR mink cell focus-forming viruses: a xenotropic-like virus, Xeno-dL, donating U3 LTR sequences, and another xenotropic-like virus or viruses providing gp70 sequences.

Murine leukemia virus-induced T-cell lymphomagenesis: integration of proviruses in a distinct chromosomal region.

A number of mink cell focus-forming (MCF) proviruses was molecularly cloned from mouse lymphoma DNA. From each clone, flanking probes were prepared to detect common integration regions in other MuLV-induced lymphomas. One clone frequently revealed variations in the molecular structure of the corresponding region (Pim-1) in other lymphomas. The results show the following. Changes in the Pim region are seen in 24 out of 93 lymphomas tested. Over 50% of the early T-cell lymphomas show integration in the Pim-1 region. The alterations are seen in different mouse strains and with various MuLVs. The observed variations are caused by the integration of predominantly MCF genomes. All integrations occur in a region spanning less than 20 kb and are associated with the transcriptional activation of a distinct region within the Pim-1 domain. The activated region does not show any homology with 13 known and three putative oncogenes.

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus.

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.

Molecular cloning and characterization of a complete DNA copy of potato spindle tuber viroid RNA.

Double-stranded cDNA has been synthesized from RNA of a severe strain of potato spindle tuber viroid using a synthetic oligodeoxyribonucleotide as a primer. Upon cloning in bacteriophage M13mp9, two recombinant phages were selected to construct a pBR322-derived plasmid containing a complete viroid DNA copy. Elucidation of the nucleotide sequence revealed four differences with the previously established sequence of another PSTV strain, three of which were base exchanges and one a deletion.

Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd.

The 6407 nucleotide-long sequence of bacteriophage M13 DNA has been determined using both the chemical degradation and chain-termination methods of DNA sequencing. This sequence has been compared with that of the closely related bacteriophage fd (Beck et al., 1978). M13 DNA appears to be only a single nucleotide shorter than fd DNA. There is an average of 3.0% of nucleotide-sequence differences between the two genomes, but the distribution of these changes is not random; the sequence of some genes is more conserved than of others. In contrast, the nucleotide sequences and positions of the regulatory elements involved in transcription, translation and replication appear to be identical in both filamentous phage DNA genomes.

The site of action of 2,4-dinitrophenol and salicylic acid upon the uncoupler-induced K+ efflux from non-metabolizing yeast.

Stimulation of K+ efflux from non-metabolizing yeast cells by 2,4-dinitrophenol or by salicylic acid occurs only after accumulation of the compounds into the cells, indicating that the site of action of the uncouplers is inside the cells. A correlation is found between the partition ratio of the lipophilic cation dibenzyldimethylammonium between cells and medium and the rate of K+ efflux.

Nucleotide sequence of the genes III, VI and I of bacteriophage M13.

A DNA region of 2750 base pairs encompassing the genes III, VI and I of bacteriophage M13 has been sequenced by the Maxam-Gilbert procedure. By establishing the nucleotide changes introduced by several amber mutations, the coding region and the regulatory signals of each gene have been deduced. The genes appear to span 1275 base pairs (gene III; mol.wt. 44,748) 339 base pairs (gene VI; mol.wt. 12,264) and 1047 base pairs (gene I; mol.wt. 39,500). Their separating non-codogenic regions are extremely short, namely two and one base pair, respectively. The C-terminal end of gene I, however, intrudes 23 nucleotides into gene IV. From the nucleotide sequence it appears that the minor capsid protein of the phage, which is encoded by gene III, is synthesized in a precursor form containing 18 extra amino acids at its N-terminal end. Furthermore, in this capsid protein two clusters of a fourfold repeat of the sequence Glu-Gly-Gly-Gly-Ser are apparent. Gene VI appears to code for a small, extremely hydrophobic polypeptide. Its total hydrophobic amino acids content of 51% suggests that this protein can only function in the host cell membrane.

Hydrolysis and synthesis of ATP by membrane-bound ATPase from a motile Streptococcus.

ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg2+ ratio of 2. Mn2+ and Ca2+ could replace Mg2+ to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide but not by sodium azide, uncouplers or bathophenanthroline. An electrochemical gradient of protons, which was artificially imposed across the membranes of Streptococcus cells by manipulation of either the K+ diffusion potential or the transmembrane pH gradient, led to ATP synthesis. ATP synthesis was abolished by proton conductors, an inhibitor of the ATPase or an increase in the extracellular K+ concentration. A comparison between the phosphate potential and the electrochemical proton gradient showed that the data found are in agreement with a stoichiometry of 2 protons translocated per molecule ATP synthesized.

Mapping of promoter sites on the genome of bacteriophage M13.

With the aid of transcription studies on restriction fragments of bacteriophage M13 DNA it has been demonstrated that at least eight promoter sites are located on the M13 genome. Five of these promoters initiate the synthesis of RNA chains which contain at their 5'-terminal end pppG (G promoters), while the other three promoters initiate RNA chains which start exclusively with pppA (A promoters). The positions of these promoter sites on the physical map are: 0.82 (G0.82), 0.88 (G0.88), 0.94 (G0.94), 0.01 (G0.01), 0.08 (G0.08), 0.36 (A0.36), 0.51 (A0.51) and 0.56 (A0.56). The G promoters were found to be clustered within a distance of one-third of the genome length from the central termination site for transcription (map position 0.77). The A promoters, however, were found at greater distances from this termination signal. Based upon the incorporation of [gamma-32P]ATP or [gamma-32P]GTP, the capacity of these promoters to initiate the synthesis of RNA chains varies. The strongest G promoters are G0.82, G0.94 and G0.08 and the strongest A promoter is A0.36. As judged from their position on the genetic map, it is postulated that two promoters, namely G0.94 and G0.01, are located within gene II. The other promoters are most probably located immediately in front of the gene VIII/VII boundary (G0.82), and immediately in front of gene V (G0.88), gene II (G0.08), gene IV (A0.36), gene I (A0.51) and gene VI (A0.56). No evidence has been obtained so far for the existence of a promoter immediately in front of gene III.