Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects

In previous minireviews in this journal, we discussed work on induction of tetrahydrobiopterin biosynthesis by cytokines and its significance for nitric oxide (NO) production of intact cells as well as functions of H4-biopterin identified at this time for NO synthases (Proc Soc Exp Biol Med 203: 1-12, 1993; Proc Soc Exp Biol Med 219: 171-182, 1998). Meanwhile, the recognition of the importance of tetrahydrobiopterin for NO formation has led to new insights into complex biological processes and revealed possible novel pharmacological strategies to intervene in certain pathological conditions. Recent work could also establish that tetrahydrobiopterin, in addition to its allosteric effects, is redox-active in the NO synthase reaction. In this review, we summarize the current view of how tetrahydrobiopterin functions in the generation of NO and focus on pharmacological aspects of tetrahydrobiopterin availability with emphasis on endothelial function.     

Linear polyethylenimine as a tool for comparative studies of antisense and short double-stranded RNA oligonucleotides

Despite the recently enlarged field of available RNA knock-down technologies, e.g., antisense oligonucleotides (ASOs) and duplexes of synthetic 21 nucleotides RNAs (siRNAs), no versatile transfection reagent has been reported to deliver different nucleic acids formats at high rates of efficiency. We have evaluated the versatility and efficacy of linear PEI in transfecting and properly delivering a broad panel of nucleic acids such as short oligonucleotides and double-stranded RNA into cells in culture.     

Effect of bradykinin analogues on the B1 receptor of rat ileum

The longitudinal muscle of isolated rat ileum is a sensitive bioassay suitable for testing compounds with antagonistic effects on the B(1) receptor. Bradykinin analogues with replacement of proline by alkyl-substituted phenylalanine at position 7 are effective on this receptor as entire molecules and have a stronger antagonistic effect than on the B(2) receptor. A corresponding desArg(9)-compound has a specific effect on the B(1) receptor and a very high antagonistic potency. [LNMPhe(2)]bradykinin as a compound without any replacement at position 7 or 8 shows antagonistic activity as well.     

Reconstructing smell

Odorant signal transduction and neurogenesis are fundamental properties of the olfactory epithelium. Many preparations have been used to elucidate some of the mechanisms underlying these properties. In this article, we briefly review these research areas and describe some of the techniques used to obtain the data. We focus specifically on the cell-culture paradigm and the data obtained from various immortal cell lines in their attempts to reconstruct the olfactory epithelium in vitro.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

Viral Determinants of Rotavirus Pathogenicity in Pigs: Evidence that the Fourth Gene of a Porcine Rotavirus Confers Diarrhea in the Homologous Host

A porcine rotavirus (prv) monoreassortant, S-F4, which carries RNA segment 4 of the pig-pathogenic variant prv 4F in the genetic background of the pig-apathogenic variant prv 4S (G. I. Tauscher and U. Desselberger, J. Virol. 71:853–857, 1997), was found to be pathogenic in gnotobiotic piglets. This indicates that RNA segment 4 of the pig-pathogenic variant prv 4F is a major determinant of pathogenicity in its homologous host.     

A symplasmic flow of sucrose contributes to phloem loading in Ricinus cotyledons

External sucrose, supplied by the endosperm in vivo, is the physiological source of sucrose for Ricinus communis L. seedlings. It is taken up by the cotyledons and exported via the sieve tubes to the growing hypocotyl and root. Two parallel pathways of external sucrose to the sieve tubes, directly via the apoplasm and indirectly after transit through the mesophyll, have already been established (G. Orlich and E. Komor, 1992). In this study, we analysed whether a symplasmic flow of sucrose contributes to phloem loading. Uptake of external sucrose into the mesophyll and into the sieve tubes, and export of total sucrose were measured with intact and exuding seedlings in the presence of p-chloromercuribenzenesulfonic acid (PCMBS). Sucrose uptake into the mesophyll and into the sieve tubes was inhibited by 80–90%. Consequently, export of total sucrose slowed down. However, after the addition of PCMBS, sucrose was transiently exported in such a high amount that could not be accounted for by the residual uptake activity nor by the amount of sucrose confined to the sieve element-companion cell complex (seccc). From the results, we conclude that most of the sucrose exported transiently had moved to the sieve tubes from a symplasmic domain larger than the seccc, comprising at least all the cells of the bundle including the bundle sheath. We suggest that the symplasmic flow of sucrose observed is a mass flow driven by a turgor pressure. As a structural prerequisite for a symplasmic flow, plasmodesmata interconnect all the cells from the bundle sheath to the sieve tubes and also occur between the bundle sheath and the mesophyll. The phloem loading pathway of Ricinus cotyledons can thus be classified as a combination of three different routes. Received: 17 October 1997 / Accepted: 9 March 1998     

YAC rescue of downless locus mutations in mice

Mice with mutations at the downless (dl) locus have defects in hair follicle, tooth, sweat gland, preputial gland, Meibomian gland, and tail development. The dl phenotype is analogous to the human genetic disorder termed autosomal hypohidrotic (or anhidrotic) ectodermal dysplasia (HED). On the basis of the identification of two related transgenic insertional mutations in the downless gene, yeast artificial chromosomes (YACs) were identified that map to the critical region of mouse Chromosome (Chr) 10. To determine which of the YACs contain the dl gene, we generated YAC transgenic mice by mouse embryo microinjections. The 200-kb YAC B25.D9 was found to rescue all of the downless defects. In addition, the transgenic YAC rescued the dominant Sleek (Dl ^slk ) allele. Since the sequences within the YAC are entirely deleted in one of the transgenic mutants, our results establish that Sleek encodes a dominant-negative protein whose effects can be reversed by expression of extra copies of the wild-type locus. Received: 26 June 1998 / Accepted: 17 July 1998     

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca²⁺ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca²⁺ and Mn²⁺ ions. We show here that addition of Mn²⁺ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca²⁺. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca²⁺-deficient media is overcome by expression of other Ca²⁺ pumps, including a SERCA-type Ca²⁺ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca²⁺ and Mn²⁺ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.     

Definition of extracellular localized epitopes of Hsp70 involved in an NK immune response

In order to define extracellular localized epitopes of Hsp70 on human tumor cells which are accessible to the immune system, six commercially available Hsp70-specific monoclonal antibodies (mAb) with different recognition sites were examined by immunological approaches. The recognition pattern of these antibodies was analyzed on purified recombinant Hsp70 proteins (rHsp70, Hsc70, DnaK), on lysates of Hsp70-expressing colon carcinoma cells (CX+) and on lysates of M21 rat-1 cells that overexpress human Hsp70 or Hsp70 fragments: ΔBgl (del 120–428) consisting of the C-terminal part and ΔSma (del 438–618) consisting of the N-terminal part of human Hsp70. All antibodies reacted equally well with rHsp70 and cytoplasmic Hsp70 derived from human tumor cells or M21 rat-1 cells. Only one antibody (MA3–007; Hsp70, Hsc70) detects a region localized within the ATPase domain of Hsp70 (amino acid 122–264) and reacts positively with the C-terminal deletion mutant ΔSma. All other antibodies, including RPN1197 are directed against the C-terminal peptide binding domain of Hsp70 and react positively with the N-terminal deletion mutant ΔBgl. Although all six antibodies detect full-length Hsp70 protein, derived from plasma membrane fractions of CX+ tumor cells, cell surface expressed Hsp70 on viable CX+ tumor cells, as determined by flowcytometry, is only recognized with the antibodies MA3–006 (Hsp70, Hsc70; 504–617), MA3–009 (Hsp70; 504–617) and RPN1197 (Hsp70). An estimation of the ratio of membrane-bound to cytoplasmic Hsp70 molecules revealed that 15–20% of total Hsp70 molecules are expressed on the plasma membrane. This tumor-selective cell surface expression of Hsp70 correlates with an increased sensitivity to lysis mediated by non-MHC restricted natural killer (NK) cells. We demonstrate that only antibodies directed against membrane-bound Hsp70 (MA3–006, MA3–009, RPN1197) inhibit NK-killing activity against Hsp70-expressing tumor cells. Taken together our data indicate that at least the C-terminal region 504–617, that contains at least one single α-helix (amino acid 512–536), has to be localized extracellularly and might be of importance for an NK-mediated anti-tumor immune response.