Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

Summary The presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the -adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.Abbreviations BPP Bovine pancreatic polypeptide - BSA Bovine serum albumin - EJP Excitatory junctional potential - FaRPs FMRFamide-related peptides - FLI FMRFamide-like immuno-reactivity - LMS Leucomyosuppressin - RIA Radioimmunoassay - RP-HPLC Reversed-phase high performance liquid chromatography - TFA Trifluoroacetic acid     

Multiple uses of liquepipets in Southern, northern, and western blots

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffers within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.     

Activation of a cryptic gene by excision of a DNA fragment.

The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene. The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation. Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation. Our model for the retention of cryptic genes (B. G. Hall, S. Yokoyama, and D. H. Calhoun, Mol. Biol. Evol. 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele. We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+. This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon. We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8). We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.     

Factors Affecting High-Oxygen Survival of Heterotrophic Microorganisms from an Antarctic Lake

We sought to determine factors relating to the survival of heterotrophic microorganisms from the high-dissolved-oxygen (HDO) waters of Lake Hoare, Antarctica. This lake contains perpetual HDO about three times that of normal saturation (40 to 50 mg liter⁻¹). Five isolates, one yeast and four bacteria, were selected from Lake Hoare waters by growth with the membrane filter technique with oxygen added to yield dissolved concentrations 14 times that in situ, 175 mg liter⁻¹. One bacterial isolate was obtained from the microbial mat beneath the HDO waters. This organism was isolated at normal atmospheric oxygen saturation. The bacteria were gram-negative rods, motile, oxidase positive, catalase positive, and superoxide dismutase positive; they contained carotenoids. The planktonic isolates grew in media containing 10 mg of Trypticase soy (BBL Microbiology Systems)-peptone (2:1) liter⁻¹ but not at 10 g liter⁻¹. Under low-nutrient levels simulating Lake Hoare waters (10 mg liter⁻¹), two of the planktonic isolates tested were not inhibited by HDO. Growth inhibition by HDO increased as nutrient concentration was increased. A carotenoid-negative mutant of one isolate demonstrated a decreased growth rate, maximal cell density, and increased cell lysis in the death phase under HDO compared with the parent strain. The specific activity of superoxide dismutase was increased by HDO in four of the five bacterial isolates. The superoxide dismutase was of the manganese type on the basis of inhibition and electrophoretic studies. The bacterial isolates from Lake Hoare possess several adaptations which may aid their survival in the HDO waters, as well as protection due to the oligotrophic nature of the lake.     

Effect of serotonin on murine macrophages: suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic receptor antagonists

Serotonin (5-HT), a mediator released from platelets at sites of inflammation, suppressed IFN-gamma-induced Ia expression in mouse bone marrow macrophages maintained in vitro. (Mean percent suppression = 63.9% +/- 9.2, n = 40.) This suppression was not toxic or endotoxin-related, was concentration-dependent, and occurred at the physiologic concentrations of 5-HT present at inflammatory sites. The concentration of 5-HT producing the half-maximal effect was 2.5 to 5.5 X 10(-8) M. Related compounds, dopamine, histamine, and tryptamine, were much less potent in suppressing IFN-gamma-induced Ia, with maximally suppressing concentrations more than 100-fold higher than the maximally suppressing 5-HT concentration. L-5-hydroxytryptophan (5-HTP), the most potent analog tested, was 10-fold less potent than 5-HT in suppressing Ia expression. The concentration of 5-HTP producing the half-maximal effect = 4 X 10(-7) M. 5-HT suppression of IFN-gamma-induced Ia expression was antagonized by the 5-HT2 type receptor antagonists spiperone, ketanserin, and LY53857. Concentrations of these agents resulting in 50% inhibition of the serotonin effect were 1.5 X 10(-8) M, 7.5 X 10(-8) M, and 4.5 X 10(-12) M, respectively. 5-HT was most effective in suppressing IFN-gamma-induced Ia when added early in culture simultaneously with IFN-gamma. These data provide functional evidence that 5-HT suppression of IFN-gamma-induced Ia expression is mediated through a 5-HT receptor with some characteristics of the 5-HT2 type. 5-HT may play a physiologic role at sites of inflammation as a modulator of the effects of IFN-gamma on macrophage function.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Chemical limnology of two artificial lakes used for both stormwater management and recreation

The aims of this study were to document the mainly chemical behaviour of two linked artificial lakes used for both stormwater management and recreation in the new town of Craigavon. Further, the understanding of their behaviour should help in their management and the design of other similar lakes.The lake mean total phosphorus (73 µg P l^–1), nitrate (0.50 mg N l^–1) and chlorophyll a (25 µg l^–1) concentrations, Secchi depth (1.2 m) and the estimated total phosphorus loading (1.98 g m^–2 a^–1) all classify the main lake as eutrophic. An important source of the phosphorus load on the lakes is the urban area of Craigavon (52% of the total load). The interrelationships between total phosphorus, chlorophyll a and Secchi depth in the main lake are similar to those in natural ones. In addition, the lake follows the total phosphorus load — trophic state relationships (lake total phosphorus and chlorophyll a concentrations and Secchi depth) found to apply elsewhere. These two points indicate that the artificial lakes in Craigavon behave similarly to natural ones.     

Subunit interactions of class I histocompatibility antigens

The kinetics of dissociation of iodinated beta 2-microglobulin (beta 2m) from the papain-solubilized class I histocompatibility antigen HLA-B7 have been investigated. In the presence of unlabeled beta 2m, most of the HLA dissociates according to a single rate constant, whereas in the absence of unlabeled beta 2m, the system approaches an equilibrium dependent upon the initial HLA concentration. When iodinated beta 2m is incubated with unlabeled HLA-B7, the rate of incorporation of beta 2m into the complex is much less dependent on the concentration than is expected for a simple association/dissociation system; instead, the system behaves as if the "activity" (in a thermodynamic sense) of the HLA heavy-chain intermediate cannot surpass a critical concentration. The dissociation rate for each class I specificity is a function of temperature, ionic strength, pH, and the status of the heavy chain (papain solubilized vs. detergent solubilized). High temperature, high ionic strength, and extremes of pH promote dissociation. The intact molecule dissociates about 10 times more slowly than the papain-solubilized molecule. In contrast, the rate of dissociation of all papain-solubilized class I antigens tested falls within the range of about a factor of 2. The presence of the carbohydrate has no effect on the rate of dissociation. The possibility that HLA class I antigen dissociation may occur in vivo within acidic internal vesicles is discussed.     

Relative developmental success of interspecific Lepomis hybrids as an estimate of gene regulatory divergence between species

The developmental success of interspecific Lepomis hybrids is used as an index of gene regulatory divergence between the green sunfish, L. cyanellus, and each of three other parental species, longear sunfish, L. megalotis, warmouth, L. gulosus, and bluegill, L. macrochirus. This gene regulatory divergence is compared to the degree of structural gene divergence among these four species (genetic distance [Nei, '78], D, ranged from 0.206 to 0.586). The developmental success of the hybrid embryos at the level of morphogenesis was higher than expected from the genetic distance between the parental species. The rates of morphogenesis of the hybrid embryos were the same as that for the green sunfish embryos. The percentage of embryos that hatched was relatively high in all crosses. However, two of the hybrid crosses resulted in enhanced percentages of hatched embryos. Slight increases in the extent of morphological abnormalities were observed in hybrids from crosses between more distantly related parental species. The schedules and levels of enzyme locus expression of the hybrids, assessed spectrophotometrically and electrophoretically for nine enzyme systems (encoded in a total of 14 loci), were different from each other and from those of the green sunfish embryos. Alterations in the time of first enzyme appearance and in the time of first increase in enzyme activity in the developing hybrid embryos were not correlated with genetic distance between parental species. However, the extents of alteration of enzyme activities over the entire period of hybrid embryogenesis were correlated with the genetic distance. We attribute the morphological and molecular anomalies observed in the hybrids to gene regulatory incompatibilities between species. Although the exact number of mutational differences and their relative developmental impacts are not known, some inferences can be drawn about the degree of divergence in gene regulation between species. It appears that an uncoupling of the rates of structural and regulatory gene evolution can occur between species of some taxa, an observation that has implications for the roles of gene regulatory differences in organismic evolution.     

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.