RESUMEN

AIMS: To determine the nucleotide sequences of adenovirus (Ad) types 1 and 6 fibre genes; to clarify the molecular basis of the distinct haemagglutination properties of subgenus C Ads and their phylogenetic relations. METHODS: Human Ad1 and Ad6 fibre genes were sequenced from genomic DNA by direct sequencing. Primer selection was based on alignment of the fibre gene of human Ad serotypes Ad2 and Ad5. Fibre based subgenus C specific polymerase chain reaction (PCR) was performed to check for deletions in field isolates of Ad6, as revealed by sequence analysis of the Ad6 prototype. A phylogenetic tree was constructed from the predicted amino acid (AA) sequences of the fibre gene of important Ads. RESULTS: Ad1 and Ad6 comprise 1746 and 1584 nucleotides, encoding 582 and 528 AA, respectively. Ad6 showed deletions in motifs 15-17 (51 AA) of the shaft when compared with Ad1, Ad2, and Ad5. Subgenus C specific PCR with both prototype and field isolates also showed deletions in Ad6. In the shaft and knob, AA homology was 58.82-72.91% and 68.89-74.59%, respectively. The tail was 100% conserved. Phylogenetically, Ad1 and Ad6, including Ad2 and Ad5, formed a subgenus specific cluster, like other serotypes. CONCLUSIONS: The fibre gene (including the knob region) of subgenus C Ads is heterogeneous, providing the molecular basis for lack of crossreactivity in the haemagglutination inhibition test. This heterogeneity could be helpful in fibre based genotyping of subgenus C field isolates. Phylogeny might be useful for subgenus specific identification of important field strains.

Asunto(s)

Adenovirus Humanos/genética , Proteínas de la Cápside/genética , Adenovirus Humanos/clasificación , Secuencia de Aminoácidos , ADN Viral/genética , Eliminación de Gen , Genes Virales , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos

RESUMEN

AIMS: To characterise a novel strain of adenovirus (Ad) type Ad8 (genome type Ad8I) involved in an epidemic keratoconjunctivitis (EKC) outbreak in Hiroshima city using serological testing and sequence analysis of the fibre and hexon gene. METHODS: A neutralisation test (NT) was performed in microtitre plates containing a confluent monolayer of A549 cells using 100 tissue culture infectious doses of virus and type specific antisera. The haemagglutination inhibition test was also carried out in microtitre plates with rat erythrocytes using four haemagglutination units of virus and twofold dilutions of serum. The fibre gene was sequenced by generating overlapping polymerase chain reaction products or by direct sequencing of genomic DNA. Primer selection was based on alignment of the fibre genes of human adenovirus serotypes Ad8, Ad19, Ad37, Ad9, and Ad15 available from Gene Bank. RESULTS: The virus strain was specifically neutralised by anti-Ad8 antibodies, although there was a major crossreaction with anti-Ad9 antibodies. Haemagglutination was equally inhibited by anti-Ad8 and anti-Ad9 antibodies. The predicted amino acid sequences of the hypervariable regions (HVRs) of the Ad8I hexon gene showed higher homology with Ad9 (83.3%) than with Ad8 (62.0%). However, the Ad8I fibre knob was more homologous to Ad8 (94.4%) than to Ad9 (91.6%). CONCLUSIONS: Ad8I is a unique strain of adenovirus because of its lower genomic homology with Ad8, major crossreactivity with Ad9 in NT, and mixed genetic organisation of HVRs of the hexon gene. These factors may have enabled the virus to circumvent acquired immunity, resulting in the outbreak.

Asunto(s)

Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/genética , Queratoconjuntivitis/virología , Infecciones por Adenovirus Humanos/epidemiología , Adenovirus Humanos/aislamiento & purificación , Animales , Secuencia de Bases , Brotes de Enfermedades , Pruebas de Inhibición de Hemaglutinación , Humanos , Japón/epidemiología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Ratas , Análisis de Secuencia de ADN

RESUMEN

AIMS: To characterise a novel strain (M86) of adenovirus (Ad) involved in epidemic keratoconjunctivitis (EKC). METHODS/RESULTS: The virus strain was neutralised by antisera to both Ad35 and Ad11. Restriction endonuclease analysis of genomic DNA showed 98% and 88% homology with Ad11 and Ad35, respectively. The deduced amino acid sequence of the hypervariable regions of (HVRs) of the hexon gene showed a higher homology with Ad35 (94.4%) than with Ad11 (83.7%). However, it was 100% homologous to Ad35 in HVRs 1, 2, 3, and 6 and to Ad11 in HVRs 4 and 6. In the fibre knob, the isolate was more homologous to Ad11 (99.4%) than to Ad35 (29.1%). CONCLUSION: This novel strain of adenovirus showed similarities with both Ad11 and Ad35. The isolation of a novel strain like Ad35+11 is important because of its association with EKC.

Asunto(s)

Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/genética , Proteínas de la Cápside/genética , Conjuntivitis Viral/virología , Queratoconjuntivitis/virología , Adenovirus Humanos/aislamiento & purificación , Adulto , Secuencia de Aminoácidos , Genes Virales , Humanos , Masculino , Datos de Secuencia Molecular , Homología de Secuencia

RESUMEN

We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , TATA Box , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis/métodos , Polarización de Fluorescencia , Sustancias Macromoleculares , Unión Proteica , Subunidades de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Fluorescencia , Proteína de Unión a TATA-Box , Factores de Transcripción/aislamiento & purificación

RESUMEN

The tryptophan synthase bienzyme complex is the most extensively documented example of substrate channeling in which the oligomeric unit has been described at near atomic resolution. Transfer of the common metabolite, indole, between the alpha- and the beta-sites occurs by diffusion along a 25-A-long interconnecting tunnel within each alphabeta-dimeric unit of the alpha(2)beta(2) oligomer. The control of metabolite transfer involves allosteric interactions that trigger the switching of alphabeta-dimeric units between open and closed conformations and between catalytic states of low and high activity. This allosteric signaling is triggered by covalent transformations at the beta-site and ligand binding to the alpha-site. The signals are transmitted between sites via a scaffolding of structural elements that includes a monovalent cation (MVC) binding site and salt bridging interactions of betaLys 167 with betaAsp 305 or alphaAsp 56. Through the combined strategies of site-directed mutations of these amino acid residues and cation substitutions at the MVC site, this work examines the interrelationship of the MVC site and the alternative salt bridges formed between Lys beta167 with Asp beta305 or Asp alpha56 to the regulation of channeling. These experiments show that both the binding of a MVC and the formation of the Lys beta167-Asp alpha56 salt bridge are important to the transmission of allosteric signals between the sites, whereas, the salt bridge between betaK167 and betaD305 appears to be only of minor significance to catalysis and allosteric regulation. The mechanistic implications of these findings both for substrate channeling and for catalysis are discussed.

Asunto(s)

Mutagénesis Sitio-Dirigida , Sales (Química)/química , Triptófano Sintasa/química , Triptófano Sintasa/genética , Alanina/genética , Regulación Alostérica/genética , Asparagina/genética , Ácido Aspártico/genética , Cationes Monovalentes/química , Deuterio/química , Dimerización , Activación Enzimática/genética , Cinética , Lisina/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Potasio/química , Compuestos de Amonio Cuaternario/química , Salmonella typhimurium/enzimología , Sodio/química , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Treonina/genética , Volumetría

RESUMEN

Studies were performed to investigate the prohormone/proprotein convertase (PC)-inhibitory properties of chemical constituents of the medicinally active plant Andrographis paniculata (AP; from the family Acanthaceae), also known as 'King of Bitters'. Among the individual components tested against the clinically important convertases, furin and PC1, neoandrographolide (a C3 O-glucoside derivative of the major constituent andrographolide) exhibited the highest inhibitory action with an IC50 of 53.5 microM against furin. The data further revealed that although andrographolide, the major bitter principle of AP, exhibited a relatively small enzyme inhibition (IC50=1.0 mM and Ki=200 microM against furin), upon succinoylation, its inhibitory action against the above convertases was enhanced significantly with a Ki in the low micromolar range (<30 microM), suggesting that a specific structural modification of the andrographolide skeleton may be exploited to develop a new class of non-peptide inhibitors of PCs. When tested against PC7, these succinoylated derivatives of andrographolide also displayed strong inhibitory action, with Ki values again in the low micromolar range. This potentially interesting observation may be attributed to the reported anti-HIV property of 14-dehydroandrographolide succinic acid monoester (DASM). It is suggested here that DASM, by virtue of this protease inhibitory property, possibly acts by suppressing the proteolytic cleavage of envelope glycoprotein gp160 of HIV, which is known to be PC-mediated, particularly by furin and PC7.

Asunto(s)

Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Diterpenos/farmacología , Subtilisinas/antagonistas & inhibidores , Succinatos/farmacología , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Ácido Aspártico Endopeptidasas/metabolismo , Diterpenos/aislamiento & purificación , Diterpenos/metabolismo , Activación Enzimática/efectos de los fármacos , Ésteres , Furina , Glucósidos/metabolismo , Glucósidos/farmacología , Humanos , Cinética , Plantas Medicinales/química , Plantas Medicinales/metabolismo , Proproteína Convertasas , Subtilisinas/metabolismo , Succinatos/metabolismo , Anhídridos Succínicos/química , Tetrahidronaftalenos/metabolismo , Tetrahidronaftalenos/farmacología

RESUMEN

Rabbit brain tryptophan hydroxylase (TPH) has been expressed in insect cells (Spodoptera frugiperda) as a histidine-tagged enzyme. The specific activity of the purified fusion enzyme is 80 nmol of 5-hydroxytryptophan/min/mg. Multifunctional regulatory 14-3-3 proteins were purified from fresh bovine brain. Phosphorylation and 14-3-3 proteins play important roles in the regulation of TPH activity. We have found that phosphorylation of TPH by cAMP-dependent protein kinase increased the activity of the hydroxylase by 25-30% and that 14-3-3 proteins increased the hydroxylase activity of phosphorylated TPH by approximately 45%. Under these conditions, the 14-3-3 proteins were not phosphorylated, and unphosphorylated TPH was not activated by 14-3-3 proteins. Surface plasmon resonance analysis demonstrated that 14-3-3 proteins bind to phosphorylated TPH with an affinity constant (Ka) of 4.5 x 10(7) M-1. Binding studies using affinity chromatography also showed that 14-3-3 proteins interact with phosphorylated TPH. The dephosphorylation of TPH by protein phosphatase-1 was inhibited by 14-3-3 proteins. Our results demonstrate that 14-3-3 proteins form a complex with phosphorylated brain TPH, thereby increasing its enzymatic activity and inhibiting its dephosphorylation.

Asunto(s)

Proteínas/metabolismo , Triptófano Hidroxilasa/metabolismo , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Animales , Encéfalo/enzimología , Bovinos , Activación Enzimática , Histidina/química , Histidina/metabolismo , Cinética , Fosforilación , Unión Proteica , Conejos , Triptófano Hidroxilasa/química , Triptófano Hidroxilasa/genética

RESUMEN

To investigate the mechanism by which the tryptophan synthase beta subunit accelerates the cleavage of the indole-3-glycerol phosphate catalyzed by the alpha subunit (alpha reaction), kinetic experiments were carried out with wild-type and mutant forms of the alpha 2 beta 2 complex. Previous studies indicate that this activation can be attributed to the conformational changes associated with the formation of a Schiff base between aminoacrylate and pyridoxal phosphate at the beta site. To test this hypothesis, we investigated a mutant form of the alpha 2 beta 2 complex having the lysine-87 of its beta subunits replaced by threonine. The mutant alpha 2 beta 2 complex (K87T) exhibits normal activity for the alpha reaction but fails to catalyze formation of L-tryptophan from L-serine and indole (beta reaction). However, the mutant enzyme can form a Schiff base intermediate with L-serine at the beta site. Using a "chemical rescue" method, we converted K87T L-serine intermediate to an aminoacrylate intermediate. Steady-state kinetic studies reveal that the aminoacrylate derivative exhibits a 7-fold enhancement in kcat/Km for the alpha reaction relative to that of the L-serine derivative of the mutant or the wild-type enzyme in the absence of L-serine. Rapid kinetic data show that the aminoacrylate derivative of the mutant enzyme exhibits a 6-fold increase in the rate constant for the indole-3-glycerol phosphate cleavage reaction. In addition, rate constants for the reverse reaction and product release steps are also altered. Together, these changes lead to a decrease in Km and an increase in kcat.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Acrilatos/metabolismo , Triptófano Sintasa/metabolismo , Regulación Alostérica , Activación Enzimática , Hidrólisis , Cinética , Mutación , Triptófano Sintasa/genética

RESUMEN

Acrylamide quenching of the tryptophan fluorescence of the lambda-repressor at different protein concentrations indicates that one of the three tryptophan residues, W129, W142, and W230, undergoes a change in environment upon self-assembly, from dimer to associated species. Quenching data suggest that this tryptophan residue is inaccessible to low concentrations of acrylamide and is blue-shifted in the associated form. In the dimer, this tryptophan residue is highly accessible to acrylamide and is red-shifted. NBS oxidation, at protein concentrations which favor the associated form, showed that this tryptophan is also significantly protected from NBS oxidation. HPLC peptide mapping of NBS-oxidized lambda-repressor, amino acid analysis, and sequencing indicate that the protected, blue-shifted tryptophan is tryptophan 230. A mutant repressor (F235C) was specifically labeled at Cys 235 with an environment-sensitive probe, acrylodan. The acrylodan fluorescence of the labeled F235C lambda-repressor undergoes a significant blue-shift, accompanied by fluorescence enhancement, upon protein association. Along with other genetic evidence, these results suggest involvement of the C-terminal tail region in the self-assembly of the lambda-repressor.

Asunto(s)

Proteínas de Unión al ADN , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Acrilamida , Acrilamidas/química , Aminoácidos/análisis , Concentración de Iones de Hidrógeno , Mapeo Peptídico , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Triptófano/química , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales

RESUMEN

This work is aimed at understanding subunit assembly in the tryptophan synthase alpha 2 beta 2 complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the beta 2 subunit of tryptophan synthase for subunit association. We utilize a mutant form of the beta 2 subunit that is unable to form the internal aldimine because lysine 87 is replaced by threonine (K87T). The K87T alpha 2 beta 2 complex is inactive in reactions catalyzed by the beta 2 subunit but retains activity in the reaction catalyzed by the alpha subunit. We find that dialysis removes pyridoxal phosphate much more rapidly from the K87T beta 2 subunit and alpha 2 beta 2 complex than from the wild type counterparts. Activity measurements, gel filtration, and subunit interchange experiments show that the alpha subunit dissociates more readily from the K87T beta 2 subunit than from the wild type beta 2 subunit. The reaction of L-serine to form an external aldimine with pyridoxal phosphate at the active site of the K87T beta 2 subunit markedly increases the affinity for the alpha subunit and slows removal of pyridoxal phosphate by dialysis. We propose that the external aldimine between L-serine and pyridoxal phosphate bridges the N-domain and the C-domain in the K87T beta 2 subunit. This interdomain bridge may mimic the internal aldimine bond in the wild type beta 2 subunit and stabilize pyridoxal phosphate binding. The interdomain bridges formed by the internal aldimine with the wild type beta 2 subunit and by the external aldimine with L-serine in the K87T beta 2 subunit may further stabilize interaction with the alpha subunit because the alpha/beta interaction site contains residues from both N- and C-domains of the beta 2 subunit.

Asunto(s)

Fosfato de Piridoxal/metabolismo , Triptófano Sintasa/metabolismo , Cromatografía en Gel , Diálisis , Estabilidad de Enzimas , Mutación , Fosfato de Piridoxal/análogos & derivados , Triptófano Sintasa/química , Triptófano Sintasa/genética

RESUMEN

The bacterial tryptophan synthase alpha 2 beta 2 complex contains an unusual structural feature: an intramolecular tunnel that channels indole from the active site of the alpha subunit to the active site of the beta subunit 25 A away. Here we investigate the role of the tunnel in communication between the alpha and beta subunits using the polarity-sensitive fluorescent probe, Nile Red. Interaction of Nile Red in the nonpolar tunnel near beta subunit residues Cys-170 and Phe-280 is supported by studies with enzymes altered at these positions. Restricting the tunnel by enlarging Cys-170 by chemical modification or mutagenesis decreases the fluorescence of Nile Red by 30-70%. Removal of a partial restriction in the tunnel by replacing Phe-280 by Cys or Ser increases the fluorescence of Nile Red more than 2-fold. A binding site for Nile Red in this region near the pyridoxal phosphate coenzyme of the beta subunit is further supported by iodide quenching and fluorescence energy transfer experiments and by molecular modeling based on the three-dimensional structure of the alpha 2 beta 2 complex. Finally, studies using Nile Red as a sensitive probe of conformational changes in the tunnel reveal that allosteric ligands (alpha subunit) or active site ligands (beta subunit) decrease the fluorescence of Nile Red. We speculate that allosteric and active site ligands induce a tunnel restriction near Phe-280 that serves as a gate to control passage of indole through the tunnel.

Asunto(s)

Conformación Proteica , Salmonella typhimurium/enzimología , Triptófano Sintasa/química , Triptófano Sintasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Codón , Gráficos por Computador , Simulación por Computador , Cisteína , Escherichia coli , Colorantes Fluorescentes , Indoles , Yoduros , Cinética , Ligandos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Oxazinas , Fenilalanina , Mutación Puntual , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , Espectrometría de Fluorescencia , Triptófano

RESUMEN

Tetramer-dimer equilibrium of lambda repressor has been studied by fluorescence anisotropy techniques. We have chosen 1-dimethylamino naphthalene-5-sulfonyl chloride (dansyl chloride)-labeled repressor to study the dissociation-association equilibrium, because of relatively long life-time of the probe (> 10 ns). Polarization of the dansyl-labeled repressor decreases with decreasing protein concentrations in the range of 20 to 0.2 microM. The decrease of anisotropy was shown to be due to reversible dissociation of the protein. Size exclusion high-performance liquid chromatography studies and polyacrylamide gel electrophoresis under native conditions (Ferguson plot) confirmed that at around 20 microM concentrations the repressor exists in predominantly tetrameric form, whereas in lower concentrations it exists in predominantly dimer form. A dissociation constant of 2.3 +/- 0.9 microM was estimated in 0.1 M potassium phosphate, pH 8.0, at 25 degrees C. A stoichiometric amount of isolated single operator shifted the tetramer-dimer equilibrium toward the dimer. Increased ionic strength had only a modest effect on the dissociation constant. The thermodynamic constants for the dissociation reaction calculated from the Van't Hoff plot was +26.6 kcal/mol for delta H and +64.7 e.u. for delta S. The rotational correlation times derived from isothermal Perrin plot indicated elongated dimers and tetramers.

Asunto(s)

Proteínas de Unión al ADN , Polarización de Fluorescencia , Proteínas Represoras/química , Factores de Transcripción/química , Biopolímeros , Compuestos de Dansilo/química , Peso Molecular , Conformación Proteica , Termodinámica , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales

RESUMEN

Urea denaturation of the lambda repressor has been studied by fluorescence and circular dichroic spectroscopies. Three phases of denaturation could be detected which we have assigned to part of the C-terminal domain, N-terminal domain and subunit dissociation coupled with further denaturation of the rest of the C-terminal domain at increasing urea concentrations. Acrylamide quenching suggests that at least one of the three tryptophan residues of the lambda repressor is in a different environment and its emission maximum is considerably blue-shifted. The transition in low urea concentration (midpoint approximately 2 M) affects the environment of this tryptophan residue, which is located in the C-terminal domain. Removal of the hinge and the N-terminal domain shifts this transition towards even lower urea concentrations, indicating the presence of interaction between hinge on N-terminal and C-terminal domains in the intact repressor.

Asunto(s)

Proteínas de Unión al ADN , Proteínas Represoras/química , Urea/farmacología , Escherichia coli/metabolismo , Cinética , Fragmentos de Péptidos/aislamiento & purificación , Conformación Proteica , Desnaturalización Proteica , Proteínas Represoras/efectos de los fármacos , Espectrometría de Fluorescencia , Compuestos de Sulfhidrilo/metabolismo , Factores de Transcripción/química , Triptófano , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales

RESUMEN

4,4'-bis(1-anilino-8-naphthalenesulfonic acid (Bis-ANS), an environment-sensitive fluorescent probe for hydrophobic region of proteins, binds specifically to the C-terminal domain of lambda repressor. The binding is characterized by positive cooperativity, the magnitude of which is dependent on protein concentration in the concentration range where dimeric repressor aggregates to a tetramer. In this range, positive cooperativity becomes more pronounced at higher protein concentrations. This suggests a preferential binding of Bis-ANS to the dimeric form of the repressor. Binding of single operator OR1 to the N-terminal domain of the repressor causes enhancement of fluorescence of the C-terminal domain bound Bis-ANS. The binding of single operator OR1 also leads to quenching of fluorescence of tryptophan residues, all of which are located in the hinge or the C-terminal domain. Thus two different fluorescent probes indicate an operator-induced conformational change which affects the C-terminal domain. The significance of this conformational change with respect to the function of lambda repressor has been discussed.

Asunto(s)

Bacteriófago lambda/metabolismo , Proteínas de Unión al ADN , Operón , Proteínas Represoras/química , Naftalenosulfonatos de Anilina , Bacteriófago lambda/genética , Sitios de Unión , Colorantes Fluorescentes , Cinética , Matemática , Conformación Proteica , Termodinámica , Factores de Transcripción/química , Tripsina , Urea , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales

RESUMEN

A new continuous coupled fluorimetric assay is described for ATPases in general. Thus phosphate released from ATP hydrolysis is coupled to the nucleoside phosphorylase reaction using 7-methylguanosine as a fluorescent substrate for the nucleoside phosphorylase reaction. The hydrolysis of 7-methylguanosine leads to 7-methylguanine, which has lower quantum yield and hence can be used to monitor ATP hydrolysis continuously. The method has the potential to be extended to GTPase and nucleotidyltransferase assays.

Asunto(s)

Miosinas/análisis , Animales , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Concentración Osmolar , Fosfatos/metabolismo , Conejos , Espectrometría de Fluorescencia

Asunto(s)

Hormona Liberadora de Gonadotropina/normas , Animales , Bioensayo , Femenino , Hormona Liberadora de Gonadotropina/farmacología , Ovulación/efectos de los fármacos , Ratas , Estadística como Asunto

RESUMEN

A simple, sensitive, and reproducible method for the detection of urinary human chorionic gonadotropin (hCG) and diagnosis of early human pregnancy is reported. A 5-ml aliquot of filtered early-morning urine sample was concentrated in a microconcentrator (M) to 0.1 ml of retentate which was diluted with 0.4 ml of distilled water and tested in a hemagglutination inhibition test (M-HIT). Also, a 0.1-ml aliquot of filtered unconcentrated urine sample was diluted with 0.4 ml of distilled water and tested in the same hemagglutination inhibition test (HIT). Urine samples from women of reproductive age; from perimenopausal, menopausal, and proteinuric women; and from adult males were tested in the HIT and M-HIT. Some of these urine samples were also tested in the mouse ovulation bioassay (MOB). The M-HIT was significantly more reliable than the HIT for diagnosis of early pregnancy 25 to 55 days after menses. Correct negative results with the M-HIT were obtained in urine samples of most of the nonpregnant cycling, perimenopausal, and menopausal women, and adult males. Urine samples from subjects with severe proteinuria gave false-positive types of reactions in the M-HIT. Positive results were obtained in the MOB with a number of urine samples from pregnant, perimenopausal, and menopausal women. A properly conducted M-HIT should be very valuable in diagnosing pregnancy as early as the 26th day of the cycle in regularly menstruating women.

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PIP: This article discusses a simple, sensitive, reproducible method for detecting HCG (human chorionic gonadotropin) in the urine and the subsequent early diagnosis of pregnancy. 5 ml of filtered urine sample (early morning) was concentrated in an M (microconcentrator) to 0.1 ml of retentate diluted with 0.4 ml of distilled water. It was then tested in a M-HIT (hemagglutination test). Another 0.1 ml aliquot of urine sample (filtered and unconcentrated) was diluted with the same amount of distilled water and tested in the same HIT (hemagglutination test). Urine samples from women of reproductive age, from perimenopausal, menopausal, and proteinuric women, and from adult males were tested in both the HIT and M-HIT, as well as in the MOB (mouse ovulation bioassay). The M-HIT Proved to be significantly more reliable than the HIT for diagnosis of early pregnancy, 25-55 days following menses. Appropriate negative results were obtained with the M-HIT in those urine samples from most of the nonpregnant, cycling, perimenopausal and postmenopausal women, and the adult males. False-positive reactions in the M-HIT resulted from the urine specimens of those with severe proteinuria. The MOB yielded positive results in a number of urine samples from pregnant, perimenopausal and menopausal women. The M-HIT, if properly done, indicates high reliability in diagnosing pregnancy as early as the 26th day in the cycles of menstruating women.

Asunto(s)

Gonadotropina Coriónica/orina , Pruebas Inmunológicas de Embarazo/métodos , Femenino , Pruebas de Inhibición de Hemaglutinación , Humanos , Embarazo , Primer Trimestre del Embarazo

RESUMEN

A simple, sensitive, and reliable single-unit nonradioactive method for the detection of human chorionic gonadotropin (hCG) in concentrated urine and the diagnosis of early pregnancy is reported. This unit, presently termed the Ayerst pregnancy test kit (APTK), consists of four components: a sampler-filter paper cone, an ultrafilter-concentrator to which a vial holder is attached, a support stand with a mirror, and an immunologic reagent vial. In the APTK, 5 to 6 ml of urine were sampled, filtered, and concentrated, and the hCG in the retentate was detected by Ayerst immunologic reagents [APTK(AY)] and by the Pregnosticon "All In" [APTK(P)]. Some of the unconcentrated urine samples (0.1 ml) were also tested in hemagglutination inhibition tests (HIT) using Ayerst [HIG(AY)] and Pregnosticon "All In" [HIT(P)] reagents. Urine samples from pregnant, nonpregnant (ovulating and nonovulating), perimenopausal, and menopausal women were tested. It was found that the APTK(AY) and APTK(P) were significantly more sensitive and reliable than the HIT(AY) and HIT(P) in detecting low levels of urinary hCG for early diagnosis of pregnancy. The sensitivity and specificity of the APTK(AY) were better than those of the APTK(P). The APTK(AY) give significantly more correct positive and negative results than the other tests performed simultaneously. The APTK(AY) is simpler and safer than the serum radioimmunoassays and radioreceptor assay presently used to detect low levels of hCG for the early diagnosis of pregnancy and other hCG-producing states.

Asunto(s)

Gonadotropina Coriónica/orina , Pruebas Inmunológicas de Embarazo/instrumentación , Estudios de Evaluación como Asunto , Femenino , Humanos , Embarazo , Pruebas Inmunológicas de Embarazo/métodos , Primer Trimestre del Embarazo

Asunto(s)

Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/farmacología , Inducción de la Ovulación/métodos , Animales , Estro , Femenino , Ratones , Embarazo , Ratas

RESUMEN

A rhythmic antifertility effect of a luteinizing hormone-releasing hormone (LH-RH) analog, [D-Ala6-des-Gly-NH210]-LH-RH-ethylamide (AY-25,205), administered intramuscularly every 3rd day staring in the afternoon of diestrus, was demonstrated in 4-day cyclic rats. The antifertility effect was achieved for a period of approximately four cycles when the females were allowed constant cohabitation with fertile males except for 24 hours following treatment. Unrestricted cohabitation resulted in some matings and pregnancies in the group treated every 3rd day and also in some of the groups treated every 4th day with restricted cohabitation. The antifertility effect of AY-25,205, with unrestricted cohabitation, disappeared when the second treatment was given 4 days after the first. It is presumed that the antifertility effect of AY-25,205 was achieved through its capacity to induce ovulation at a physiologically "wrong time" (i.e., 1 day before the expected day of proestrus) and through its effect on mating behavior. The present experimental models suggest that AY-25,205 or similar analogs could be potentially useful for a more reliable rhythm method of birth control in humans, by timing ovulation and narrowing the fertile period of the cycle.

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PIP: The rhythmic antifertility effect of an LH-RH analog, AY-25,205, was investigated in 4-day cyclic rats. All groups (10 animals in each) received 20-120 ng/rat im between 3:00 and 3:30 P.M. the day of diestrus. Treatment continued for either 2 or 4 consecutive cycles, and subsequent injections were given either on the 3rd or 4th consecutive day; cohabitation was either unrestricted or prevented for 24 hours posttreatment. Only 1 rat of 40 in the 3-day. restricted cohabitation groups became pregnant during the 1st cycle, and the antifertility effect continued. However, for those groups treated every 4th day and allowed unrestricted cohabitation, 25 of 30 animals became pregnant the night of the 2nd injection. It is suggested that the antifertility effect of AY-25,205 is due to an ability to induce ovulation 1 day before the appearance of gonadotropin-induced lordosis. AY-25,205 or similar analogs may have application in humans by making more precise the time of ovulation, which would permit more effective use of the "rhythm method" of birth control.

Asunto(s)

Fertilidad/efectos de los fármacos , Hormona Liberadora de Gonadotropina/análogos & derivados , Conducta Sexual Animal/efectos de los fármacos , Animales , Estro , Femenino , Masculino , Ovulación/efectos de los fármacos , Embarazo , Ratas