A comparison of the in vitro activities of amodiaquine and desethylamodiaquine against isolates of Plasmodium falciparum.

The antimalarial activities of amodiaquine, the desethyl metabolite of amodiaquine, chloroquine, and mefloquine were evaluated against 35 field isolates of Plasmodium falciparum collected from eastern Thailand, October-December 1985, to define patterns of cross-resistance among these compounds. The assay system was based on the in vitro inhibition of schizont maturation. The parasites were generally sensitive to mefloquine (mean 50%-inhibitory concentrations = 9.

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study.

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine.

Activation of protein kinase C and L calcium channels enhances binding of biotinylated corticotropin-releasing hormone by anterior pituitary corticotropes.

Arginine vasopressin (AVP) potentiates corticotrope responses to CRH by increasing the percentage of target cells that secrete in a reverse hemolytic plaque assay for ACTH. The present studies were designed to test more specific effects of AVP and its second messengers on CRH binding to individual corticotropes. Spectrophotometric analyses of 560 corticotropes from fractions enriched to 90% by counterflow centrifugation showed a 30% increase in the average area of the dark blue label for biotinylated CRH after a 1-h exposure to 10 nM AVP or after activation of protein kinase-C [by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or L calcium channels (by Bay K 8644).

Enrichment of corticotropes by counterflow centrifugation.

Anterior pituitary corticotropes represent only 9-10% of the mixed pituitary cell population. However, their small size precludes their enrichment because they cannot be separated from the more abundant PRL and GH cells. They can be induced to enlarge by adrenalectomy, and this report describes the separation of larger CRH-responsive corticotropes from a subpopulation of small pituitary cells.

Synaptic transmission between dissociated adult mammalian neurons and attached synaptic boutons.

In most studies of synaptic currents in mammalian central neurons, preparations have been used in which synaptic currents are recorded at some distance from the synapse itself. This procedure introduces problems in interpretation of the kinetics and voltage-dependent properties of the synaptic current. These problems have now been overcome by the development of a preparation in which presynaptic vesicle-containing boutons have been coisolated with the soma of individual neurons, thus providing the opportunity to study synaptic currents under conditions of both adequate voltage control and internal ionic perfusion.

Changes in the number of GnRH-receptive cells during the rat estrous cycle: biphasic effects of estradiol.

The number of gonadotropin-releasing hormone (GnRH) receptors is known to vary throughout the estrous cycle and in other endocrine states in the rat. These changes in receptors parallel closely the concentrations of serum estradiol during the cycle. In the present study, we used two different cytochemical techniques to determine if changes in GnRH receptors represented alterations in the number of GnRH-receptive cells.

A histone H1 protein in sea urchins is encoded by a poly(A)+ mRNA.

Typical histone genes lack intervening sequences and encode small mRNAs (400-800 nucleotides) with short leader and trailer regions. Most histone mRNAs are not polyadenylylated but rather terminate in a highly conserved stem and loop structure. The early, late, and testis-specific histone genes of sea urchins, described to date, have this typical histone gene structure.

Differential storage and release of luteinizing hormone and follicle-releasing hormone from individual gonadotropes separated by centrifugal elutriation.

Two subtypes of pituitary gonadotropes are known to exist: monohormonal and multihormonal. When separated by centrifugal elutriation monohormonal cells are found in the small fractions whereas multihormonal cells predominate in the large gonadotrope-enriched fractions. Since GnRH is known to shift the proportion of subtypes to cells that are mainly multihormonal, we tested its effect on the small monohormonal gonadotropes.

Characterization of the structure and transcriptional patterns of the gene encoding the late histone subtype H1-beta of the sea urchin Strongylocentrotus purpuratus.

We have cloned and characterized the gene encoding the late histone H1-beta subtype from the sea urchin Strongylocentrotus purpuratus. The gene contains all of the upstream sequence homologies previously seen in late H1-gamma genes. The expression of H1-beta mRNA is coordinated with that of H1-gamma mRNA, and like H1-gamma it is expressed in all adult somatic tissues tested.

A novel method to demonstrate parathyroid hormone binding on unfixed living target cells in culture.

We present a rapid and specific procedure that enables one to identify living target cells of peptide hormones within heterogeneous cell populations by means of morphological demonstration of ligand-receptor binding. This is exemplified using a biotinylated parathyroid hormone (PTH) analogue (biotinyl-b-PTH 1-84) which reacts with avidin-fluorescein (avidin-FITC) as a histochemical marker. The experiments revealed a fine dot-like distribution pattern of binding after 1-5 min of incubation at room temperature, changing to a more clustered pattern after 10 min of incubation.

Both basal and ontogenic promoter elements affect the timing and level of expression of a sea urchin H1 gene during early embryogenesis.

Late histone H1-beta mRNA accumulates with the correct ontogenic pattern following microinjection of the cloned gene into fertilized sea urchin eggs. Sequences upstream of the gene encoding the sea urchin H1-beta protein contain both basal and developmentally regulated elements. One late H1-specific activator sequence (USE IV) is required for the accumulation of mRNA following the blastula stage of development.

In vitro activity of pyronaridine against field isolates and reference clones of Plasmodium falciparum.

Pyronaridine, a 9-substituted 1-aza-acridine, was assayed for in vitro activity against clinical and field isolates as well as characterized clones of Plasmodium falciparum. The in vitro antimalarial activity of pyronaridine was compared to activities of standard antimalarials against multidrug-resistant isolates of P. falciparum from eastern and northern Thailand using an assay based on the inhibition of schizont maturation.

Evaluation of an in vitro assay system for drug susceptibility of field isolates of Plasmodium falciparum from southern Thailand.

An in vitro assay system has been developed to evaluate the susceptibility of field isolates of Plasmodium falciparum to standard and new antimalarials. The assay used drugs which were serially diluted in the field and determined effective drug concentrations by quantitating schizont maturation after a variable incubation period. Based on the ID50 values, a series of isolates from Yala in southern Thailand were shown to be resistant to chloroquine (187 nM) but only moderately resistant to amodiaquine (23.

Analysis of dose-response curves for the in vitro susceptibility of Plasmodium falciparum to antimalarials using a pocket computer.

A small pocket computer, Sharp PC-1500, was programmed to analyze the dose-response curves generated by the in vitro assay of antimalarials against field isolates of Plasmodium falciparum. Using nonlinear regression the analysis provided an estimate of the 50% inhibitory concentration with 95% confidence limits and a graph of the data points and regression function line. The pocket computer with the nonlinear regression program offers a powerful, portable, and inexpensive data analysis system suitable for field use.

In vitro mefloquine resistance of Plasmodium falciparum isolated from the Burmese border region of Thailand.

The in vitro susceptibility of twenty isolates of Plasmodium falciparum from Tha Song Yang, Tak province, Thailand were determined. The isolates were resistant to chloroquine (IC50 = 220 nM; MIC = 762 nM), quinine (IC50 = 252 nM; MIC = 1010 nM), and pyrimethamine (IC50 = 16400 nM; MIC = 43100 nM) but generally sensitive to mefloquine (IC50 = 6.90 nM; MIC = 20.

Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe.

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH.

Cytological factors that support nonparallel secretion of luteinizing hormone and follicle-stimulating hormone during the estrous cycle.

Gonadotropes from cycling female rats were studied to investigate possible mechanisms for the nonparallel release of LH and FSH. The percentages of total gonadotropes increased from 14% during estrus (E) to 18% by diestrous day 2. More of these cells became multihormonal on the morning of proestrus (P; from 46% during diestrus to 69%).

Molecular characterization of the histone gene family of Caenorhabditis elegans.

The core histone genes (H2A, H2B, H3 and H4) of Caenorhabditis elegans are arranged in approximately 11 dispersed clusters and are not tandemly arrayed in the genome. Three well-characterized genomic clones, which contain histone genes, have one copy of each core histone gene per cluster. One of the clones (lambda Ceh-1) carries one histone cluster surrounded by several thousand base-pairs of non-histone DNA, and another clone (lambda Ceh-3) contains a histone cluster duplication surrounded by non-histone DNA.

Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures.

Intact anterior pituitary tissue and primary anterior pituitary cultures were stained with 1:30,000 anti-TRH and 1:10,000 anti-GnRH using the peroxidase antiperoxidase immunocytochemical technique. Stains applied to serial ultrathin sections of intact pituitaries showed that TRH immunoreactivity could be localized in secretory granules of thyrotropes, gonadotropes and corticotropes whereas GnRH immunoreactivity was found only in gonadotropes and corticotropes. Long-term primary pituitary cultures were studied to remove the anterior pituitary cells from hypothalamic influences.

Involvement of sodium channels and two types of calcium channels in the regulation of adrenocorticotropin release.

Recent electrophysiological studies from this laboratory demonstrated that anterior lobe corticotropes exhibited a tetrodotoxin-sensitive sodium current and two types of voltage-dependent calcium currents, consisting of low threshold (transient) and high threshold (long lasting) components. The present report describes cytophysiological and cytochemical studies that used specific blockers of each of these currents to assess their role in the regulation of CRF binding and ACTH secretion and storage. Two dihydropyridines, nimodipine and the pure antagonist enantiomer (-)R202-791, which block high threshold Ca2+ channels, decreased 1 h basal release by 54-74% and CRF-mediated (5 min or 3 h) release completely.

Membrane currents of identified isolated rat corticotropes and gonadotropes.

Membrane currents of identified, isolated corticotropes and gonadotropes from mammalian anterior pituitary gland have been evaluated. Pituitary gonadotropes and corticotropes were isolated enzymatically and stained in the living state using biotinylated gonadotropin-releasing hormone (Bio-GnRH) or biotinylated corticotropin-releasing hormone (Bio-CRF) followed by avidin fluorescein. Electrophysiological recordings were made with patch-clamp electrodes in the whole-cell clamp configuration.

Secretion from corticotropes after avidin-fluorescein stains for biotinylated ligands (CRF or AVP).

Viable pituitary target cells were identified with biotin-labeled corticotropin-releasing factor (1-Bio-CRF) or arginine vasopressin (Bio-AVP) and avidin-fluorescein (cell sorter grade). Stain was reduced from 8-10% of pituitary cells to less than 3% if nonbiotinylated ligands were added to compete with their respective biotinylated ligand for specific binding sites. However, AVP did not decrease the stain for 1-Bio-CRF nor did CRF decrease the Bio-AVP stain.

Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: effect of gonadotropin-releasing hormone.

The cytosolic concentration of free Ca2+ ([Ca2+]i) in normal rat pituitary cells separated by centrifugal elutriation was monitored with the fluorescent Ca2+ indicator Quin 2. GnRH (10(-7) M) induced a rapid rise (6-8 sec) in the gonadotroph's [Ca2+]i, followed by a plateau phase of prolonged elevated [Ca2+]i which lasted about 15 min. The stimulatory effect of GnRH was dose dependent, with an ED50 of 10(-9) M, and was blocked by the potent antagonist [Dp-Glu1,pclPhe2,DTrp3.

Use of the reverse hemolytic plaque assay to study the regulation of anterior lobe adrenocorticotropin (ACTH) secretion by ACTH-releasing factor, arginine vasopressin, angiotensin II, and glucocorticoids.

A reverse hemolytic plaque assay (RHPA) for ACTH was developed to study the responses of anterior lobe corticotropes to secretagogues-CRF, arginine vasopressin (AVP), angiotensin II (A-II), or to a 6- to 24-h pretreatment with corticosterone. Tests showed that optimal plaque formation was obtained after 3-4 h with 1:100-1:200 anti ACTH-(25-39) and 1:20-1:50 complement. Under optimal basal conditions, 6.

Isolation, characterization, and expression of the gene encoding the late histone subtype H1-gamma of the sea urchin Strongylocentrotus purpuratus.

We cloned and characterized the gene encoding H1-gamma, a late histone subtype of the sea urchin species Strongylocentrotus purpuratus. The predicted primary sequence of H1-gamma is 216 amino acids in length and has a net charge of +70, which is high for a somatic H1 histone. The H1-gamma gene appears to be a unique sequence gene that is not tightly linked to the core histone genes.