Particular RNA primer from growth medium differentially stimulates in vitro DNA synthesis and in vivo cell growth of Neurospora crassa and its slime mutant

Summary Purine rich small RNA-primer molecules (about 10–12 nucleotides), secreted into the growth medium of 3-h germinated conidia of N. crassa, strongly stimulated a concentration-dependent in vitro DNA synthesis of N. crassa slime mutant as well as DNAs from the human cancer cells but did not affect that from normal cells. These RNA-primer molecules stimulated also in vivo cell growth of N. crassa slime mutant, but not of the N. crassa wild type. Our studies suggest that DNAs from the slime mutant of N. crassa as well as DNAs from human cancerous cells provide increased sites for enhanced in vitro and in vivo replication of DNAs. RNA-primer molecules can be hydrolyzed by T1 RNase but not by pancreatic RNase.     

The β-tubulin gene of Epichloë typhina from perennial ryegrass (Lolium perenne)

Summary Epichloë typhina is a biotrophic fungal pathogen which causes choke disease of pooid grasses. The anamorphic state, Acremonium typhinum, is placed in the section Albo-lanosa along with related, mutualistic, seeddisseminated endophytes. As an initial study of gene structure and evolution in Epichloë and related endophytes, the -tubulin gene, tub2, of the perennial ryegrass choke pathogen (EtPRG) was cloned and sequenced. The coding sequence and the predicted -tubulin amino acid sequence were highly homologous to the Neurospora crassa homologs, and to one of the two -tubulin genes of Emericella nidulans. However, two introns characteristic of the N. crassa and Em. nidulans genes were absent in the E. typhina gene. Furthermore, one of the remaining introns possessed the uncommon 5 splice junction, GC. In contrast to published observations concerning other Ascomycetes, a mutant of EtPRG, selected for resistance to methyl-2-benzimidazole carbamate (benomyl), possessed no alteration of its -tubulin coding sequence.     

Chromosomal mapping of an alcC disruption with respect to amdA in Aspergillus nidulans

Summary We report the use of the riboB gene for a gene replacement in the alcC gene of Aspergillus nidulans, and show by reverse genetics that the alcC gene is very closely linked to the amdA gene.     

Oral Administration of Gammalinolenic Acid, an Unsaturated Fatty Acid with Anti-inflammatory Properties, Modulates Interleukin-1β Production by Human Monocytes

Administration of gammalinolenic acid (GLA), an unsaturated fatty acid, reduces joint inflammation in patients with rheumatoid arthritis. Addition of GLA in vitro suppresses release of interleukin-1 (IL-1) from human monocytes stimulated with lipopolysaccharide (LPS). LPS-induced IL-1 release is followed by IL-1-induced IL-1 release, an amplification process termed autoinduction. We show here, using IL-1 stimulation to simulate autoinduction, that administration of GLA to healthy volunteers and to patients with inflammatory arthritis reduces LPS-induced IL-1 secretion mainly by reducing autoinduction of IL-1. GLA reduces LPS-induced pro-IL-1 mRNA modestly and IL-1-induced pro-IL-1 gene expression markedly. In addition to reducing amplification of IL-1, GLA increases the amount of IL-1 receptor antagonist (IL-1Ra) secreted from stimulated cells, thereby facilitating an increase in the secreted IL-1Ra/IL-1 ratio. IL-1 is important to host defense, but the amplification mechanism may be excessive in genetically predisposed individuals. Thus, reduction of IL-1 autoinduction may be protective in some patients with endotoxic shock and with diseases characterized by chronic inflammation.     

Serum bactericidal activity of two newer quinolones againstSalmonella typhi compared with standard therapeutic regimens

The bactericidal activity of two newer quinolones, ciprofloxacin and ofloxacin, against eight strains of Salmonella typhi was examined by the serum dilution test and studies of bacterial killing kinetics in human serum, and compared to standard regimens. Bactericidal titers for ciprofloxacin ranged from 1388 to 1119 two hours and from 1119 to 157 six hours after volunteers received an oral dose of 500 mg. The respective titers obtained after a 200 mg oral dose of ofloxacin were somewhat lower, but still exceeded 116 in all instances. Studies of bacterial killing kinetics demonstrated a rapid bactericidal action of both drugs against all strains tested. Compared to the classical anti-typhoid agents chloramphenicol, cotrimoxazole and amoxicillin, the new quinolones showed both markedly higher bactericidal titers and more rapid killing of Salmonella typhi in human serum.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

Summary Embryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar Chinese Spring have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of Chinese Spring calli is closely related to that of the initial Chinese Spring calli, while non-embryogenic subcultures derived from non-embryogenic fragments of Chinese Spring calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar Aquila. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (Talent, Thésée and Capitole) display mitochondrial DNA arrangements similar to those found in cultivar Aquila. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.Abbreviations mtDNA mitochondrial DNA - ctDNA chloroplast DNA - rRNA ribosomal RNA - kb kilobase pair - cv cultivar - 2,4-D 2,4-dichlorophenoxyacetic acid     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

Molecular size-dependent leakage of intracellular molecules from frog skeletal muscle fibers permeabilized with β-escin

The permeability of -escin-treated cell membrane was characterized in terms of the permeant molecular size, by monitoring the leak of cytoplasmic molecules in frog skeletal muscle fibers. With a low concentration of -escin (5 M), most of the cellular ATP was lost within 30–40 min (as revealed by rigor force generation), whereas a fluorescence-labeled dextran injected into the cytoplasm (10 kDa) and cytoplasmic proteins (14–80 kDa) slowly leaked out of the cell. A high concentration of -escin (50–100 M) accelerated the leak of large molecules. Therefore, low concentrations of -escin may be used as a means of permeabilizing the cell membrane to relatively small molecules, while retaining a major fraction of the cellular macromolecules.Abbreviations MOPS 3-[N-morpholino]propanesulfonic acid - KMS potassium methanesulphonate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - EGTA ethylene glycol-bis( -amino-ethyl ether)N,N,N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Absence of long-term modulation of ventilation by dead-space loading during moderate exercise in humans

The stability of arterial PCO₂ (P_aCO₂) during moderate exercise in humans suggests a CO₂-linked control that matches ventilation (_E) to pulmonary CO₂ clearance (CO₂). An alternative view is that _E is subject to long-term modulation (LTM) induced by hyperpnoeic history. LTM has been reported with associative conditioning via dead-space (V_D) loading in exercising goats (Martin and Mitchell 1993). Whether this prevails in humans is less clear, which may reflect differences in study design (e.g. subject familiarisation; V_D load; whether or not _E is expressed relative to CO₂; choice of P_aCO₂ estimator). After familiarisation, nine healthy males performed moderate constant-load cycle-ergometry (20 W-80 W-20 W; <lactate threshold, _L): day 1, pre-conditioning, n=3; day 2, conditioning (V_D=1.59 l, doubling _E at 20 W and 80 W), n=8 with 10 min rest between tests; and, after 1 h rest, post-conditioning, n=3. Gas exchange was determined breath-by-breath. Post-conditioning, neither the transient [phase 1, phase 2 (1, 2)] nor steady-state _E exercise responses, nor their proportionality to CO₂, differed from pre-conditioning. For post-conditioning trial 1, steady-state _E was 28.1 (4.7) l min^–1 versus 29.1 (3.8) l min^–1 pre-conditioning, and mean-alveolar PCO₂ (a validated P_aCO₂ estimator) was 5.53 (0.48) kPa [41.5 (3.6) mmHg] versus 5.59 (0.49) kPa [41.9 (3.7) mmHg]; the 1 _E increment was 4.2 (2.9) l min^–1 versus 5.2 (1.9) l min^–1; the 2 _E time-constant () was 64.4 (24.1) s versus 64.1 (25.3) s; _E/CO₂ was 1.12 (0.04) versus 1.10 (0.04); and the _E-CO₂ slope was 21.7 (3.4) versus 21.2 (3.2). In conclusion, we could find no evidence to support ventilatory control during moderate exercise being influenced by hyperpnoeic history associated with dead-space loading in humans.     

β subunits determine the time course of desensitization in rat α3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of 34 or 32 mRNAs. Currents of several A amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n1.7) with a half-maximal activation concentration (EC₅₀) of 15–30 M. In 34 receptors, ACh displayed cooperativity (n=1.8) but was less efficacious than DMPP, yet its EC₅₀ was about equal to that of DMPP. Finally, in 32 receptors, ACh was much less efficacious and had a much lower EC₅₀. Desensitization induced by either DMPP or ACh was slow in 34 nicotinic ACh receptors but was rapid and extensive in 32 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.     

Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges

This study focuses on recent improvement in epithelial monolayer cultures originating from whole extirpated Botryllus schlosseri (Urochordata) buds. Buds (n = 2,000) were taken at different (A to D) blastogenic stages. We tested the suitability of 35 combinations of various substrates and media on attachment, cell spread, epithelial growth frequencies and on monolayer lifespans. Under favorable conditions, cultured buds at blastogenic stages B to D (but not stage A) started to attach to the substrates following a 3-day transient period that leads to formation of spheres and attached monolayers. Substrate type is important for the attachment and the development of monolayers. Under various culture conditions, some of stages B and C buds develop (3–20 days) one or more large (1 mm diameter) spheres. Stage D buds develop monolayers (up to 20% of buds) without going through a sphere phase. Neither spheres nor attached monolayers of epithelium were observed in stage A bud cultures. Spheres grew at a rate of 60 m in diameter per day using specific medium types and did not attach unless the appropriate substrate was present. When attached, epithelial monolayers expanded at a rate of 200 m in diameter per day, for 3–15 days, and subsequently detached and died. Sixteen types of media were tested. Medium and substrate combinations were found to determine epithelial lifespan. These results revealed significant improvements in the culture of epithelial monolayers from Botryllus palleal buds. However, an early senescence of the developed epithelial sheets (up to two weeks from onset of appearance) may indicate an internal ageing clock that should be taken into consideration in future approaches.     

An efficient cell-free translation system from Aspergillus nidulans and in vitro translocation of prepro-a-factor across Aspergillus microsomes

Summary We describe the preparation of an in vitro translation system from heat shock-treated Aspergillus nidulans, capable of supporting efficient and faithful synthesis of proteins from natural and in vitro transcribed eukaryotic messages. In vitro synthesized prepro--factor was translocated across Aspergillus nidulans microsomal membranes in either the homologous A. nidulans or a yeast cell-free system. The translocated prepro--factor was protected from digestion by protease and glycosylated to higher MW forms.     

Improved transformation efficiency of Aspergillus niger using the homologous niaD gene for nitrate reductase

Summary Aspergillus niger transformation frequencies of up to 1,176 transformants per g DNA were achieved using the plasmid vector pSTA10 containing the A. niger nitrate reductase structural gene. Analysis of genomic endonuclease cleaved DNA from nitrate utilising transformants by DNA hybridisation, showed that most integration events are as a result of homologous recombination. The niaD transformation system was used successfully for the introduction of the unselected Escherichia coli fusion genes lacZ, encoding -galactosidase, and uidA, for -glucuronidase, as well as the Neurospora crassa tub-2 gene, for -tubulin. pSTA10 was also capable of transforming niaD mutants of other filamentous fungi such as A. nidulans, A. oryzae and Penicillium chrysogenum.     

Cultivar variability for the presence of plastid DNA in pollen of Pisum sativum L.: implications for plastid transmission

Summary The mode of plastid transmission in the garden pea (Pisum sativum L.) was analyzed cytologically using the DNA-fluorochrome 4,6-diamidino-2-phenylindole (DAPI) in conjunction with epifluorescence microscopy. The reproductive cells of mature pollen obtained from 12 inbred lines and cv Early Alaska were examined for the presence or absence of DAPI-stained plastid DNA aggregates. Plastid DNA was detected in all 13 pea lines examined, although there was variability with regard to the percentage of pollen graines showing plastid DNA aggregates of generative cells (ranging from 3% in accession 82-12r to 65% in accession 82-14n). These cytological results may indicate genetic variability for plastic DNA inheritance in the garden pea. This paper is dedicated to the memory of Gerald A. Marx     

Amino-acid alterations in the β-tubilin gene of Neurospora crassa that confer resistance to carbendazim and diethofencarb

We have previously shown that increased sensitivity to diethofencarb in the carbendazim(MBC)-resistant F914 strain of Neurospora crassa is caused by a single amino-acid change in -tubulin, ¹⁹⁸Glu to Gly. Three diethofencarb-resistant mutants that are also resistant to MBC were isolated from strain F914. They contained single base-pair-substitution mutations in the -tubulin gene. The amino acid changes in -tubulin, Phe from ²⁵⁰Leu, Val from ¹⁶⁵Ala, and Ala from ²³⁷Thr, were responsible for diethofencarb-resistance in the mutant strains FR511, FR513, and FR421, respectively. The amino acid at position 198 of -tubulin in these mutants was Gly, which is the same as in strain F914. -tubulin genes with ¹⁹⁸Glu were constructed by site-directed mutagenesis. The altered -tubulin genes derived from FR511 and FR421 transformed the wild-type strain to resistance to MBC, indicating that ²⁵⁰Phe and ²³⁷Ala in -tubulin are responsible for resistance not only to diethofencarb but also to MBC.     

Wild chromosomal variants inAspergillus nidulans

Pulsed-field gel electrophoresis and a chromosome-specific cosmid DNA library were used to determine the karyotypes of wild-typeAspergillus nidulans isolates from around the world. Overall, little structural variation was found, with a few major exceptions. One isolate possessed a non-essential B-chromosome of about 1.0 million base pairs (mb). Another isolate had undergone a non-reciprocal translocation of about 1.6 mb of chromosome VI onto chromosome VIII. Other than these chromosomal differences, these isolates appeared phenotypically normal. To analyze its effects on meiosis, the translocation isolate was outcrossed with another wild-type derivative that had a normal electrophoretic karyotype. This cross produced a range of phenotypes, including duplicated progeny that had a barren phenotype similar to that described forNeurospora partial disomics. The duplication was somewhat vegetatively unstable. This is the first association of sterility with chromosomal duplication inA. nidulans.     

Cloning and characterization of the three enzyme structural genes QUTB,QUTC and QUTE from the quinic acid utilization gene cluster in Aspergillus nidulans

Summary Heterologous DNA probes from the quinic acid gene cluster (QA) in Neurospora crassa (Schweizer 1981) have been used to isolate the corresponding gene cluster (QUT) from Aspergillus nidulans cloned in a phage vector. N. crassa probes for each of the three enzyme structural genes in the cluster have been used to identify the corresponding genes within the A. nidulans cloned DNA. The three genes are in the same relative sequence [dehydrogenase (1), QA-3 QUTB; dehydratase (3), QA-4 QUTC; dehydroquinase (2), QA-2 = QUTE] though contained within a 3.4 kb DNA sequence in Aspergillus compared to a 5.4 kb sequence in Neurospora.The A. nidulans dehydroquinase (2) gene QUTE has been shown to complement an auxotrophic mutantaro D6 of Escherichia coli lacking biosynthetic dehydroquinase when tested for growth at 30 °C.A mutant of A. nidulans lacking catabolic dehydroquinase (2) and designated qutE208 has been isolated and shown to be tightly linked to the gene cluster, which maps between the ornB and fwA loci in linkage group VIII.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Regional sequence homologies in starch-degrading enzymes

The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by -, - and glucoamylases (-amylases), cyclodextrinases, -glucosidases, and debranching enzymes. Saccharomyces cerevisiae cannot utilize starch. Our laboratory has previously co-expressed the Bacillus amyloliquefaciens -amylase (AMY) and the Saccharomyces diastaticus glucoamylase (STA2) genes in S. cerevisiae. A gene encoding a debranching enzyme (pullulanase) from Klebsiella pneumoniae ATCC15050 was cloned and its nucleotide sequence determined. This gene will be co-expressed with the - and -amylase to produce an amylolytic S. cerevisiae strain. Extensive data base comparisons of the K. pneumoniae pullulanase amino-acid sequence with the the amino-acid sequences of other debranching enzymes and -, - and -amylases (from bacteria, yeasts, higher fungi and higher eukaryotes), indicated that these debranching enzymes have amino-acid regions similar to those found in -amylases. The conserved regions in -amylases comprise key residues that are implicated in substrate binding, catalysis, and calcium binding and are as follows. Region 1: DVVINH; region 2: GFRLDAAKH and region 4: FVDNHD. When comparing conserved regions, no similarity could be detected between debranching enzymes and - and -amylases. Present address: M.P.I. für Biophysikalische Chemie, Postfach 2841, D-3400 Göttingen, Germany (until 31 Dec 1993)