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At least 271 records · Page 15Linked to original sources
PubMed1998

Nuleclear extracts of Crithidia fasciculata contain a factor(s) that binds to the 5'-untranslated regions of TOP2 and RPA1 mRNAs containing sequences required for their cell cycle regulation.

The Crithidia fasciculata replication protein A gene, RPA1, and topoisomerase II gene, TOP2, encode proteins involved in the replication of nuclear and mitochondrial DNA, respectively. Transcripts of both genes accumulate periodically during the cell cycle and attain their maximum levels just before S phase. Octamer consensus sequences within the 5'-untranslated region (UTR) of both genes have been shown to be necessary for cycling of these transcripts. Using a gel retardation assay, we show here that nuclear extracts of C. fasciculata contain a protein factor(s) that binds specifically to RNA from 5'-UTRs of TOP2 and RPA1 genes. In addition, mutations in the consensus octamer sequence abolish binding to the RNA in both cases. Ultraviolet cross-linking using a radiolabeled TOP2 5'-UTR probe identified proteins with apparent molecular masses of 74 and 37 kDa in the RNA-protein complex. Nuclear extracts prepared from synchronized cells show that the binding activity varies during the cell cycle in parallel with TOP2 and RPA1 mRNA levels. These results suggest that the cell cycle regulation of the mRNA levels of trypanosomatid DNA replication genes may be mediated by binding of specific proteins to conserved sequences in the 5'-UTR of their transcripts.

Animals↗
PubMed2005

Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186.

Positive transcription elongation factor b (P-TEFb) regulates eukaryotic gene expression at the level of elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein, HEXIM1 or HEXIM2. To further understand how P-TEFb is regulated, we analyzed the stoichiometry of all the known components of the large, inactive P-TEFb complex. Mutational analyses of a putative coiled coil region in the carboxyl-terminal portion of HEXIM1 revealed that the protein is a dimer in solution and remains a dimer after binding to 7SK. Although a HEXIM1 dimer contains two potential RNA binding motifs and ultimately recruits two P-TEFb molecules, it associates with only one molecule of RNA. The first 172 nucleotides of the 330-nucleotide 7SK are sufficient to bind HEXIM1 or HEXIM2, and then recruit and inhibit P-TEFb. Deletion of the first 121 amino acids of HEXIM1 allowed it to inhibit P-TEFb partially in the absence of 7SK RNA. Mutation of a conserved tyrosine (Tyr(271) in HEXIM1) to alanine or glutamate or mutation of a conserved phenylalanine (Phe(208)) to alanine, aspartate, or lysine, resulted in loss of inhibition of P-TEFb, but did not affect formation of the 7SK.HEXIM.P-TEFb complex. Analysis of T-loop phosphorylation in Cdk9 indicated that phosphorylation of Thr(186), but not Ser(175), was essential for kinase activity and for recruitment of P-TEFb to the 7SK.HEXIM complex. A model illustrates what is currently known about how HEXIM proteins, 7SK, and P-TEFb assemble to maintain an activated kinase in a readily available, but inactive form.

Amino Acid Sequence↗
PubMed1984

Cytotoxicity of a cell-reactive immunotoxin containing ricin A chain is potentiated by an anti-immunotoxin containing ricin B chain.

In vitro killing of the human Daudi cell line by either univalent [F(ab')] or divalent (IgG) forms of rabbit anti-human Ig (RAHIg) coupled to ricin A chain can be specifically potentiated by a "piggyback" treatment with ricin B chain coupled to goat anti-rabbit Ig (GARIg). When cells are treated with univalent immunotoxin (IT) [F(ab') RAHIg-A] and then cultured, IT can be detected on the cell surface for at least 5 h, since GARIg-B can still enhance killing at this time. These results provide a strategy for in vivo use of A chain- and B chain-containing IT.

Adjuvants, Immunologic↗
PubMed1989

Observations of some oxygen-containing and sulfur-containing organic molecules in cold dark clouds.

Observations of nine oxygen- and sulfur-containing organic molecules have been made toward the cold dark clouds TMC-1 and L134N. We have confirmed the presence of para-ketene (H2C2O) in TMC-1, have for the first time observed ortho-ketene, and find a total ketene column density approximately 1 x 10(13) cm-2. Thioformaldehyde (H2CS) is easily detectable in both TMC-1 and L134N, with a column density about 5 times larger in the former source (approximately 3 x 10(13) cm-2). The fractional abundance of ketene is comparable to the predictions of ion-molecule chemistry, while that of thioformaldehyde in TMC-1 is one to two orders of magnitude greater than that expected from such models at steady state. Interstellar sulfur chemistry thus continues to be poorly understood. We set upper limits for the column densities of formic acid (HCOOH), vinyl alcohol (CH2CHOH), methyl formate (HCO2CH3), formamide (NH2CHO), methyl mercaptan (CH3SH), isothiocyanic acid (HNCS), and thioketene (H2C2S) in both sources.

Astronomical Phenomena↗
PubMed2005

Differential immunogenicity of a DNA vaccine containing the Streptococcus mutans wall-associated protein A gene versus that containing a truncated derivative antigen A lacking in the hydrophobic carboxyterminal region.

Two plasmid DNA constructs were obtained by cloning separately into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO the wall-associated protein A (wapA) gene of Streptococcus mutans GS-5 or its truncated derivative antigen A (agA) gene encoding a known candidate antigen for dental caries vaccine. The immunogenicity of the two constructs, designated pcDNA-wapA and pcDNA-agA, was compared by intranasal immunization of two groups of mice using the cationic DMRIE-C (1,2-dimyristyloxypropyl-3-dimethylhydroxy ethyl ammonium bromide-cholesterol) as an adjuvant. Immunization with pcDNA-wapA or pcDNA- agA resulted in specific salivary IgA and systemic IgG antibodies to the target antigens after two doses given at 3-week intervals. Higher salivary IgA level was observed in the mice immunized with the pcDNA-wapA vaccine compared to those immunized with the pcDNA-agA vaccine. Furthermore, anti-WapA antibody inhibited S. mutans sucrose-dependent adherence suggesting a potential protection against S. mutans colonization of the tooth, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs in addition to those found in AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated that full-length WapA is a better candidate vaccine antigen than the soluble AgA, which is truncated in the hydrophobic membrane and wall-spanning region.

Animals↗
PubMed1997

The chick type III collagen gene contains two promoters that are preferentially expressed in different cell types and are separated by over 20 kb of DNA containing 23 exons.

Type III collagen is present in prechondrogenic mesenchyme, but not in cartilages formed during endochondral ossification. However, cultured chick chondrocytes contain an unusual transcript of the type III collagen gene in which exons 1-23 are replaced with a previously undescribed exon, 23A; this alternative transcript does not encode type III collagen. This observation suggested that, although production of type III collagen mRNA is repressed in chondrocytes, transcription of the type III collagen gene may continue from an alternative promoter. To test this prediction, we isolated and characterized both the upstream and internal promoters of this gene and tested their ability to direct transcription in chondrocytes and skin fibroblasts. The upstream promoter is active in fibroblasts, but inactive in chondrocytes, indicating that repression of type III collagen synthesis during chondrogenesis is transcriptionally mediated. Additionally, sequences in intron 23, preceding exon 23A, function as a highly active promoter in chondrocytes; transcription from this promoter is repressed in fibroblasts. Thus transcriptional control of the type III collagen gene is highly complex, with two promoters separated by at least 20 kb of DNA that are preferentially expressed in different cell types and give rise to RNAs with different structures and functions.

Animals↗
PubMed1983

The preparative separation of sialic acid-containing lipids from sulphate group-containing glycolipids from small intestine of different animals. Analysis by thin-layer chromatography and detection of novel species.

Acidic glycolipids (gangliosides and glycolipid sulphates) were purified from non-glycolipid contaminants by silicic acid chromatography of their acetylated derivatives. Acetylation converted gangliosides into non-polar neutral and weakly acidic derivatives. These were separated from acetylated sulphate-containing glycolipids by chromatography on DEAE-cellulose. The fractions thus isolated from small intestine of 7 different animals were analyzed by thin-layer chromatography. This revealed a considerable species-related variation of both gangliosides and sulphoglycolipids. Mass spectrometry demonstrated a novel ganglioside in guinea-pig small intestine and a novel sulphoglycolipid in mouse small intestine.

Acetylation↗
PubMed1999

Diverse effects of tyrosine kinase inhibitors on follicle-stimulating hormone-stimulated estradiol and progesterone production from rat granulosa cells in serum-containing medium and serum-free medium containing epidermal growth factor.

Epidermal growth factor (EGF) has been shown to influence FSH-stimulated estradiol (E2) and progesterone (P4) production from granulosa cells. RG 50810, a tyrosine kinase inhibitor (TKI), has previously been shown to inhibit the EGF-receptor tyrosine kinase. RG 50810 has also been shown to inhibit FSH-stimulated increases in mRNA for steroidogenic enzymes, implying a functional role of tyrosine kinases in FSH action in granulosa cells. However, inhibition of FSH-stimulated steroidogenesis by TKIs has not been evaluated in connection with the effects of EGF in granulosa cells. In the present studies, FSH-stimulated E2 production was inhibited similarly by inhibitors of protein kinase A (H-89) and protein kinase C (calphostin C) and by TKIs, and none of the inhibitors were capable of reversing the EGF-induced inhibition of FSH-stimulated E2 production. FSH-stimulated P4 production was enhanced dramatically in serum-containing medium with concentrations of TKI that were near previously reported IC50s. The enhancing effect of TKIs was less evident in serum-free medium. Addition of EGF to serum-free medium enhanced FSH-stimulated P4 production, and the TKIs reversed EGF-enhanced P4 production, but in a manner similar to that of protein kinase A inhibitor H-89. Compared to results in serum-free medium, the potency of RG 50810 and genistein to inhibit the effects of EGF on P4 production was 3- to 8-fold greater relative to H-89. These studies have demonstrated that TKIs RG 50810 and genistein selectively inhibit the effects of EGF on FSH-stimulated P4 production in granulosa cell cultures. In contrast, these studies have demonstrated nonselective inhibition of FSH-stimulated E2 and P4 production by TKIs in serum-free medium, in which it is not clear which enzyme system is affected by the compounds tested.

Animals↗
PubMed1990

Stochastic properties of ion channel openings and bursts in a membrane patch that contains two channels: evidence concerning the number of channels present when a record containing only single openings is observed.

If a single ion channel record is observed in which two ion channels are never simultaneously open, then it is often of interest to know whether the observations indeed arose from the activity of only one ion channel. This question can be answered if it is possible to calculate the distribution of the duration of runs of single openings in a membrane patch that contains two active channels. If the observed run of single openings is much longer than that expected for a patch with two channels it is likely that only one channel was active. An approximate method is presented for calculating the distribution of the duration of runs of single openings in a patch with two active channels; this method has the advantage that it can be calculated from observable quantities, and requires no knowledge of the details of the ion-channel mechanism or its rate constants. The accuracy of this approximation is tested by exact calculations of the properties of runs of single openings, and of single bursts, for two specific mechanisms and a large range of rate constants. The approximation is good in all cases in which openings occur singly, or in closely spaced bursts. If, as is common in practice, openings occur in clusters that are separated by long shut periods, then overlap of clusters from two different channels may be detected, if no double opening is produced, as a period in the middle of a cluster in which the probability of being open doubles. The results derived here can be applied to such a period to test whether it results from the simultaneous activity of two channels, rather than from a change in the properties of a single channel.

Animals↗
PubMed1997

Human genital tissues containing DNA of adeno-associated virus lack DNA sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus DNA.

The detection of DNA of the helper virus-dependent adeno-associated virus type 2 (AAV-2) in biopsies of material from spontaneous abortion and in tissue samples from the uterus raises the question of whether sequences of known helper viruses can be detected simultaneously within the same specimen despite the lack of histological evidence for the presence of lytic viruses. Therefore, we performed PCR analyses with primers detecting DNA sequences of viruses (adenovirus, herpes simplex virus and human cytomegalovirus) known for their helper activity in the replication of adeno-associated viruses. In addition, PCR was performed to detect DNA of human papillomaviruses (HPV), which were recently shown to be able to help AAV replication in vitro. In no cases were sequences of the known helper viruses found. However, HPV DNA was detected in approximately 60% of paraffin sections from uterus biopsies and cervical lesions containing AAV DNA and in approximately 70% of material from early miscarriage. This finding suggests that HPV may be a helper virus for AAV.

Abortion, Spontaneous↗
PubMedDate not supplied

Study on the comparative immunogenicity of a recombinant DNA hepatitis B vaccine containing pre-S components of the HBV coat protein with non pre-S containing vaccines.

A new recombinant hepatitis B vaccine (SCI-B-VAC), derived from Chinese hamster ovary (CHO) cells and consisting of both the major S protein and the minor pre-S1 and pre-S2 proteins of the viral coat were compared with two yeast-derived vaccines containing only S proteins (B-Hepavac II and Engerix-B) for immunogenicity in human volunteers in a randomized controlled study. Two hundred and ninety-five healthy subjects completed the 12 month follow up. There was no difference in the mean age and sex distribution among the three study groups. Seroconversion rates for all the three groups were similar at months 6, 9 and 12. However, hepatitis B surface antibody (anti-HBs) geometric mean titres (GMT) were significantly higher with 10 micrograms SCI-B-VAC and 20 micrograms Engerix-B than with 10 micrograms B-Hepavac-II at months 6, 9 and 12. SCI-B-VAC at month 6 also showed a significantly higher anti-HBs GMT than Engerix-B (295 vs 143 miu/mL, P < 0.02).

Adolescent↗
PubMed1995

Expression of the genes for insecticidal crystal proteins in Bacillus thuringiensis: cryIVA, not cryIVB, is transcribed by RNA polymerase containing sigma H and that containing sigma E.

To investigate the mechanism of transcriptional regulation of cryIVA and cryIVB, encoding 130-kDa dipteran-active crystal proteins, in Bacillus thuringiensis subsp. israelensis, we introduced each gene into several sporulation mutants of Bacillus subtilis. A spoIIG mutation, the wild-type gene of which encodes sigma E precursor, completely blocked the cryIVB transcription. In contrast, low but detectable transcription of cryIVA was observed in the spoIIG mutant. In the wild-type B. subtilis, no transcription of cryIVB was detected before T2 (2 h after the onset of stationary phase), while the cryIVA transcription started at the late exponential phase at low levels. Furthermore, in a wild-type strain of B. thuringiensis subsp. israelensis, transcription of cryIVA began earlier than that of genes encoding other crystal components, cryIVB and cytA. A consensus sequence recognized by an RNA polymerase containing sigma H of B. subtilis was found upstream of the transcription start point of cryIVA, which overlapped with that recognized by sigma E.

Bacillus subtilis↗
PubMed1995

The effect of a commercially available triclosan-containing toothpaste compared to a sodium-fluoride-containing toothpaste and a chlorhexidine rinse on 4-day plaque regrowth.

Many compounds could be added to toothpaste to assist plaque inhibition, but ionic interactions can cause formulation difficulties. Moreover, the actual chemical action of a plaque inhibitory agent added to a toothpaste is difficult to assess when the product is used in the conventional manner, i.e., in addition to toothbrushing. The non-ionic antimicrobial triclosan has been incorporated in toothpastes and shown to have variable plaque inhibitory activity both alone and in conjunction with certain polymers or metal ions. Little is known of the efficacy of triclosan toothpastes compared to conventional fluoride toothpastes. The aim of this study was to compare a commercially available toothpaste containing 0.3% triclosan/co-polymer with a sodium fluoride toothpaste for chemical plaque inhibitory effects over a 4-day period. The study was designed to stratify the relative efficacy plaque inhibitory action of the products, comparisons were made with a positive control, chlorhexidine rinse and a negative control, saline. The study design was a randomised single blind crossover design balanced for first-order carryover. A total of 18 healthy, dentate volunteers participated in the study. On day 1 of each period the volunteers suspended toothcleaning and rinsed 2 x daily with the allocated mouthrinse or toothpaste slurry. On day 5, the plaque on the teeth was disclosed and scored by index and area. Increasing plaque scores were in the order chlorhexidine, triclosan toothpaste, fluoride toothpaste, and saline. Chlorhexidine was significantly more effective than all the other agents tested, and both toothpaste preparations were significantly better than the saline rinse. There was no significant difference between the two toothpaste rinses. Consistent with other studies the triclosan toothpaste offers only moderate plaque inhibitory properties when compared to a conventional toothpaste.

Adolescent↗
PubMed1998

A dual-signaling mechanism mediated by the ArcB hybrid sensor kinase containing the histidine-containing phosphotransfer domain in Escherichia coli.

The two components ArcB and ArcA play a crucial role in the signal transduction implicated in the complex transcriptional regulatory network that allows Escherichia coli to sense various respiratory growth conditions. ArcB is a hybrid sensor kinase having multiple phosphorylation sites in its primary amino acid sequence, including a transmitter, a receiver, and a histidine-containing phosphotransfer (HPt) domain. ArcA is a DNA-binding transcriptional regulator with a receiver domain. Results of recent in vitro studies revealed multistep His-to-Asp phosphotransfer circuitry in the ArcB-ArcA signaling system. For this report we conducted a series of in vivo experiments using a set of crucial ArcB mutants to evaluate the regulation of the sdh operon. The results suggested that the phosphorylated His-717 site in the HPt domain of ArcB is essential for anaerobic repression of sdh. Nonetheless, the ArcB mutant lacking this crucial His-717 site does not necessarily exhibit a null phenotype with respect to ArcB-ArcA signaling. The HPt mutant appears to maintain an ability to signal ArcA, particularly under aerobic conditions, which results in a significant repression of sdh. Based on these and other in vivo results, we propose a model in which ArcB functions in its own right as a dual-signaling sensor that is capable of propagating two types of stimuli through two distinct phosphotransfer pathways.

Bacterial Outer Membrane Proteins↗
PubMed1984

Formation of cytoplasm-containing vesicles from double-walled coated invaginations containing oligodendrocytic cytoplasm at the axon-myelin sheath interface in adult mammalian central nervous system.

Double-walled coated invaginations ( DWCIs ) of the axolemma, containing oligodendroglial cytoplasm and presumably giving rise to double-walled coated vesicles ( DWCVs ), are observed in the central nervous system (CNS) of adult monkey and mouse. By means of serial sections, DWCIs are clearly distinguishable from the also observable adaxonal invaginations of oligodendroglial cytoplasm. DWCIs range in diameter from 60 to 190 nm with a circular or oval cross section. Their incidence in different regions of the CNS has been determined and evidence is adduced that DWCIs occur randomly along the axolemma.

Animals↗
PubMed1987

Biopharmaceutical characteristics of a suppository base containing poly (oxyethylene)-poly (oxypropylene) copolymer, Unilube. I. Effects of a suppository base containing Unilube 70DP-950B on release and rectal absorption of aminopyrine in rabbit.

The rectal absorption of aminopyrine (AP) from a three-component-system suppository base (TCS), which is a mixture of oleaginous base, water-soluble base and the poly-(oxyethylene) poly-(oxypropylene) block copolymer, Unilube, was investigated. Albino rabbits were used as the animal model. The bioavailability of oleaginous base and water-soluble base (polyethylene glycol 4000) were 44.3% and 82.6%, respectively. The time to reach maximum plasma concentration (Tmax) of the oleaginous and the water-soluble bases were 21.0 +/- 8.2 min and 33.0 +/- 16.4 min, respectively. When TCS was used, its Tmax was 30 min and increasing the amount of Unilube in the base from 5% (w/w) to 20% (w/w) decreased the bioavailability from 100.4% to 47.2%. The softening temperature of TCS was 55-57 degrees C. Collectively, TCS containing 5% of Unilube gave significantly higher bioavailability than the other bases without softening until 57 degrees C. Also, the results of the two different release tests were compared with the results of an animal experiment in this paper.

Animals↗
PubMed1999

Ribonuclease-gold labels proteoglycan-containing cytoplasmic granules and ribonucleic acid-containing organelles--a survey.

An enzyme-affinity-gold method to detect RNA in routinely prepared ultrastructural samples is based on the affinity of the gold-coupled enzyme, ribonuclease, for its substrate, RNA. High concentrations of a known inhibitor of RNase, heparin, are uniquely located in human mast cell granules. Specific labeling for the presence of heparin in these structures was determined using the RNase-gold (R-G) reagent based on the RNase inhibitor property of heparin. This property was used to probe for the presence of proteoglycans (PG) known to be present in a wide variety of ultrastructural samples, none of which contain heparin. In addition to known subcellular sites of RNA, the R-G reagent was shown to bind to PG-rich cytoplasmic granules in a wide variety of leukocytes and secretory cells of epithelial, endocrine, and neuroendocrine origin. This newly recognized property was used to image the changing distribution of labeled PGs during cellular maturation, secretion, and recovery from secretion of secretory cells in vivo, ex vivo, in vitro and in isolated, biochemically defined guinea pig basophil granule preparations.

Animals↗
PubMed1989

Different responses of albumin-containing and alpha-fetoprotein-containing cells to estriol in the adult mouse liver.

A single intraperitoneal injection of 10 mg estriol (E3) rapidly induced a transient decrease in the serum albumin (ALB) level in 3 to 5 days in adult mice. At a dose of 5 mg, E3 showed a remarkable threshold effect on the decline of serum ALB. By immunohistochemistry, two types of ALB-positive cells were found in the liver. Type 1 cells were stained intensely and almost homogeneously throughout all or nearly all their entire cytoplasm, and type 2 cells showed only granular or vesicular cytoplasmic deposits. After administration of 10 mg E3, the type 1 cells diminished rapidly and markedly in direct proportion to the fall in serum ALB from the centro- and mediolobular zones, where they had been normally localized either singly or in small groups. Simultaneously, the granular or vesicular deposits that had been normally stained very weakly in the type 2 cells in the centro- and mediolobular zones became intensely stained along with cells in the perilobular zone. Alpha-fetoprotein (AFP)-containing cells in the liver appeared in maximal numbers at 5 to 7 days after E3 administration. These serological and cytological changes after E3 administration are here discussed on the basis of a possible inverse relationship between ALB and AFP gene expression.

Albumins↗