Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.

Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re-accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady-state Ca2+ concentration was obtained using Sr2+, a well-known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3-fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10-fold drop in a few minutes.     

Pro-Thyrotropin-Releasing Hormone Processing by Recombinant PC1

Abstract: Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH₂), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [³H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH_25–152 or preproTRH_25–158) and a 10-kDa C-terminal peptide (preproTRH_154–255 or preproTRH_160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (preproTRH_25–76 or preproTRH_25–82) and a 3.8-kDa peptide (preproTRH_83–108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (preproTRH_206–255). The optimal pH for these cleavages was 5.5. ZnCl₂, EDTA, EGTA, and the omission of Ca²⁺ inhibited the formation of pYE₂₇ (preproTRH_25–50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.     

Analysis of active nucleolus organizing regions in Capsicum (Solanaceae) by silver staining

Nucleolar activity of 22 samples belonging to nine diploid species of Capsicum was analyzed in somatic metaphases and interphase nuclei. They are: C, chacoënse, C. parvifolium, C. frutescens, C. chinense, C. annuum var. annuum, C. baccatum var. pendulum, C. pubescens, all with 2n = 24, and C. mirabile var. mirabile and C. campylopodium with 2n = 26. Silver staining was applied for the first time in Capsicum, providing useful markers for chromosome identification in combination with other banding techniques already employed in the genus. From two to eight AgNORs (silver-stained nucleolus organizing regions) were found in the diploid complement of the taxa studied. Nucleolar organizers are located at secondary constrictions of chromosomes which are conventionally stained or banded (C-banding or fluorochrome banding). Polymorphism of AgNORs and attached satellites often occurs. Nucleoli are usually fused to a variable extent. Number and position of active rDNA loci are variable not only between but also within species and populations. Homologies in position of NORs between species were established. The data obtained are related to previous conclusions on phylogenetic relationships in Capsicum. Possible trends of karyotype evolution concerning nucleolar organizers are discussed, and four NORs in the diploid complement (on chromosome pairs #1 [m] and #12 [st]) are regarded as the plesiomorphic condition.     

PCR for bioaerosol monitoring: sensitivity and environmental interference.

The PCR technique has potential for use in detection of low concentrations of airborne microorganisms. In this study, the sensitivity of PCR and its susceptibility to environmental interference were assessed with Escherichia coli DH1 as the target organism. Air samples, containing environmental bioaerosols, were collected with AGI-30 samplers and seeded with E. coli DH1 cells. Parallel studies were performed with cells seeded into the sampler prior to collection of air samples to determine the effects of environmental inhibition and sampling stress on the PCR assay. Baseline studies were also performed without environmental challenge or sampling stress to compare two protocols for cell lysis, solid phase and freeze-thaw. Amplification of a plasmid target sequence resulted in a detection limit of a single bacterial cell by the freeze-thaw and solid-phase methods within 5 and 9 h, respectively. With a genomic target, the sensitivity of the solid-phase method was 10-fold lower than that of freeze-thaw. Samples which contained 10(3) to 10(4) CFU of environmental organisms per m3 inhibited amplification; however, a 1/10 dilution of these samples resulted in successful amplifications. No difference in sensitivity of the PCR assay was obtained as a result of sampling stress, although a 10-fold decrease in culturability was observed. A field validation of the protocol with genomic primers demonstrated the presence of airborne E. coli and/or Shigella spp. in outdoor samples. This study indicates that the PCR method for detection of airborne microorganisms is rapid and sensitive and can be used as an alternative method for air quality monitoring.     

Effect of probenecid on 5-hydroxyindoleacetic acid in cisternal cerebrospinal fluid of rats with portacaval anastomosis

Portal-systemic encephalopathy (PSE) is characterized by a neuropsychiatric disorder progressing through personality changes, to stupor and coma. Previous studies have revealed alterations of serotonin and of its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in brain tissue and CSF in experimental (rat) and human PSE. Increased brain 5-HIAA concentrations could result from its decreased removal rather than to increased serotonin metabolism. In order to evaluate this possibility, CSF 5-HIAA concentrations were measured using an indwelling cisterna magna catheter technique at various times following end-to-side portacaval anastomosis in rats (the most widely used animal model of PSE) treated with probenecid, a competitive inhibitor that blocks the active transport of acid metabolites out of the brain and CSF. Following portacaval anastomosis and probenecid treatment, CSF concentrations of 5-HIAA were increased to a greater extent than in sham-operated controls. When data were expressed as per-cent baseline values, the relative increase of CSF 5-HIAA in portacaval shunted rats following probenecid treatment was not significantly different from sham-operated controls. These findings confirm that increased 5-HIAA in the CNS in experimental PSE results from increased 5HT metabolism or turnover and that the probenecid-sensitive acid metabolite carrier is intact in PSE.     

Mating type differentiation in tetrahymena thermophila: Characterization of the delayed refeeding effect and its implications concerning intranuclear coordination

Mating type determination in Tetrahymena thermophila involves developmentally programmed, heritable alterations of the macronucleus, localized to the mtd locus. This determination can be predictably controlled by the environmental conditions during macronuclear development, eg, temperature and time of refeeding. In this article we have further characterized the effects of delayed refeeding on mating type determination, as revealed by the frequency of mating types among the progeny of a cross. Our results show that 1) the magnitude of this starvation effect decreases with temperature of conjugation and becomes undetectable at 18°C; 2) starvation during the interval 14 to 22 hr (after conjugation is induced at 30°C) is a necessary and sufficient condition for the induction of starvation effects; 3) relative mating type frequencies vary monotonically with nutrient concentration present during this critical period; and 4) sister macronuclei, developing under starvation conditions in the same cytoplasm, differentiate majority mating types characteristic of early or late refeeding; sister macronuclei show no apparent correlation with each other. On the basis of our observations on early and late refed cells, we propose that the composition of the newly developed macronucleus is the outcome of two key events: 1) mating type determination at the mtd locus and 2) differential molecular cloning of generally one or two autonomously replicating fragments (ARFs) of the macronuclear DNA bearing the mtd locus.     

Effects of electron donors on Ca2+-dependent K+ transport in one-step inside-out vesicles from the human erythrocyte membrane

The interactions between reducing agents and Ca2+ in the activation of Ca2+-dependent K+ transport have been studied in one-step inside-out vesicles. The artificial electron donor system ascorbate + phenazine methosulphate increases the apparent sensitivity to Ca2+ by about 5-times over control values (half activation constant, about 5 X 10(-8) M) while oxidized cytochrome c decreases the sensitivity to about 1/3 of the controls. Using redox buffers at a fixed pCa it is shown that the shift from the low to the high-affinity state can be accounted by the reduction of a membrane component accepting two electrons and with an apparent standard redox potential (pH 7.5) of 47 mV. The electrons can be transferred directly from reduced PMS or to oxidized cytochrome c, but not from ascorbate, NADH or reduced glutathione.     

The taxonomy of the family Paramphistomidae Fischoeder, 1901 with special reference to the morphology of species occurring in ruminants IV. Revision of the genusGigantocotyle Näsmark, 1937 and elevation of the subgenusExplanatum Fukui, 1929 to full generic status

Summary The genusGigantocotyle Näsmark, 1937 is redefined and restricted to contain four valid species, namely,G. gigantocotyle (Brandes in Otto, 1896) Näsmark, 1937 (type species);G. formosanum (Fukui, 1929) Näsmark, 1937;G. symmeri Näsmark, 1937 andG. duplicitestorum Näsmark, 1937.G. lerouxi Yeh, 1957 is regarded as a synonym ofG. symmeri Näsmark, 1937.The subgenusExplanatum Fukui, 1929 is redefined and raised to full generic rank to contain three valid species, namely,E. explanatum (Creplin, 1847) Fukui, 1929;E. bathycotyle (Fischoeder, 1901) Yamaguti, 1958 andE. anisocotylea (Faust, 1920) Yamaguti, 1958.Paramphistomum siamense Stiles & Goldberger, 1910 andP. fraternum Stiles & Goldberger, 1910 are considered synonyms ofExplanatum explanatum (Creplin, 1847) Fukui, 1929.The valid species are redescribed and illustrated and scanning electron microphotographs of the tegumental surfaces of some species are provided. A key to the species of each genus is given.Part of a thesis approved by the University of London for the award of the Ph.D. degree.Part of a thesis approved by the University of London for the award of the Ph.D. degree.     

Irrigation planning to transform subsistence agriculture: Lessons From El Salvador

Experience with irrigation schemes designed to improve the productivity of subsistence farmers in Latin America seems to parallel the results of the green revolution in Southeast Asia. Although in some cases the impact on the productivity has been significant, in far too many cases the pervasive pattern of inequity has been reinforced. This case study of the Irrigation District of Zapotitan in El Salvador analyses some of the reasons for the unexpected results, with particular reference to the planning process. It essentially illustrates that the transformation of subsistence agriculture through irrigation demands new institutions designed not only to service agricultural production but also to ensure the accrual of benefits on the targeted group.