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Synthetic oligonucleotide hybridization probes to diagnose hop stunt viroid strains and citrus exocortis viroid.

Four species of synthetic oligonucleotide probes for the diagnosis of hop stunt viroid (HSV) and citrus exocortis viroid (CEV) were devised. Probe HSV-1 detected all the members of HSV group, such as HSV-hop, HSV-grapevine, HSV-cucumber, HSV-citrus and a viroid-like RNA isolated from plum trees affected by plum dapple fruit disease. Probe HSV-2 discriminated HSV-grapevine from the other members of HSV group. HSV-hop and HSV-grapevine consist of the same numbers of nucleotides, with only one nucleotide exchange. It was also shown that the two viroids were indistinguishable by their biological and physicochemical properties. However, by using probe HSV-2, HSV-hop and HSV-grapevine were apparently differentiated. Probe CEV-1 detected all the members of potato spindle tuber viroid (PSTV) group, such as PSTV, CEV and chrysanthemum stunt viroid (CSV). Probe CEV-2 discriminated CEV from the other members of PSTV group. It is thus emphasized that synthetic oligonucleotide probes are useful for the diagnosis of viroids and their related strains. It was discussed that the method can be used for the diagnosis of viruses and their related strains.

Base Sequence↗

The hopping response after two-stage cortical ablation in young and adult rabbits.

In a group of 14 rabbits aged between 14 and 21 days a hemidecortication was performed in the left hemisphere. A normal monopedal lateral hopping response developed in both forelegs. At adult age, 13 weeks after the first lesion, the remaining cortex on the right side was ablated. Three days afterwards the hopping response was tested again. In all animals, the hopping response remained positive in the right foreleg, contralateral to the lesioning at early age. However, in 10 out of 14 animals the response was absent on the left side, contralateral to the recent lesion. These results show that the hopping response is not mediated by corticofugal connections of the remaining hemisphere of the foreleg contralateral to the early lesion.

Age Factors↗

Electronic energy levels and hopping conductivity of an entire molecule of hagfish insulin.

The quantum-chemical calculation of an entire molecule of hagfish insulin was done by the ENFC method in which the matrix elements were calculated at the ab initio level using a minimal basis set with simulation of the aqueous solution environment. The ac conductivity for hagfish insulin was also calculated at the ab initio level by random walk theory. All the results were compared with those of pig insulin. It is shown that the reduction of HOMOs and LUMOs localized on the active sites of hagfish insulin agrees with the decrease in the biological reactivity of the insulin. The analysis of primary hopping events showed that a different sequence could influence the biological activity of insulin through the distribution of the hopping centers and the quantities of the hopping frequencies. The curve of the frequency versus ac conductivity of hagfish insulin shows that the different amino acid sequences of proteins influence the hopping conductivity. However, the electronic properties of native proteins are dominated by the three-dimensional conformations. Finally, the electronic mechanism of trans-membrane signal transforms by insulin and its receptor, which had been proposed by Ye and Ladik, was clearly described.

Amino Acid Sequence↗

Differential design for hopping in two species of wallabies.

Hindlimb musculoskeletal anatomy and steady speed over ground hopping mechanics were compared in two species of macropod marsupials, tammar wallabies and yellow-footed rock wallabies (YFRW). These two species are relatively closely related and are of similar size and general body plan, yet they inhabit different environments with presumably different musculoskeletal demands. Tammar wallabies live in relatively flat, open habitat whereas yellow-footed rock wallabies inhabit steep cliff faces. The goal of this study was to explore musculoskeletal differences between tammar wallabies and yellow-footed rock wallabies and determine how these differences influence each species' hopping mechanics. We found the cross-sectional area of the combined ankle extensor tendons of yellow-footed rock wallabies was 13% greater than that of tammar wallabies. Both species experienced similar ankle joint moments during steady-speed hopping, however due to a lower mechanical advantage at this joint, tammar wallabies produced 26% more muscle force. Thus, during moderate speed hopping, yellow-footed rock wallabies operated with 38% higher tendon safety factors, while tammar wallabies were able to store 73% more elastic strain energy (2.18 J per leg vs. 1.26 J in YFRW). This likely reflects the differing demands of the environments inhabited by these two species, where selection for non-steady locomotor performance in rocky terrain likely requires trade-offs in locomotor economy.

Animals↗

Regulation of osteoblastic phenotype and gene expression by hop-derived phytoestrogens.

Certain plant-derived compounds show selective estrogen receptor modulator (SERM) activity and may therefore be an alternative to the conventional hormone replacement therapy, which prevents osteoporosis but is also associated with an increased risk of breast and endometrial cancers. In the current study, we tested the effects of the hop-derived compounds 8-prenylnaringenin, 6-prenylnaringenin, xanthohumol and isoxanthohumol (1) to modulate markers of differentiation and gene expression in osteoblasts and (2) to regulate proliferation in MCF-7 breast cancer cells. Additionally, we analyzed the ER-binding affinities of these hop compounds as well as the ER-mediation of their effects. Bone-forming activity and ER-subtype specificity were investigated by measuring alkaline phosphatase (AP) activity in hFOB/ERalpha cells and regulation of gene transcription for AP, interleukin-6, pS2 and von Willebrand factor (VWF) in U-2 OS/ERalpha and U-2 OS/ERbeta cells. Our results demonstrate that AP, pS2 and VWF mRNA levels are significantly increased by the compounds in an estrogen-like manner via both ERalpha and ERbeta, while IL-6 is down-regulated in U-2 OS/ERalpha cells. Consistently, AP enzymatic activity is up-regulated by all compounds in hFOB/ERalpha9 cells. Depending on their concentration, all compounds show proliferative effects in MCF-7 cells. Except for 8-PN the hop constituents display an ERbeta-preference. Reversal of estrogen-specific AP-induction in Ishikawa cells indicates an ER-regulated mechanism. Finally, the flavonoids display cytotoxic effects only at high concentrations (> or =10(-4)M). In summary, we have demonstrated for the first time that specific phytoestrogen compounds found in hop extracts exert estrogen-like activities on bone metabolism. Regarding a potential for use in osteoporosis-prevention therapy, the dosage of a phytoestrogen, which is taken, will play an important role concerning a desired in vivo profile.

Adult↗

Xanthohumol and related prenylflavonoids from hops and beer: to your good health!

Xanthohumol (3'-[3,3-dimethyl allyl]-2',4',4-trihydroxy-6'-methoxychalcone) is the principal prenylated flavonoid of the female inflorescences of the hop plant ('hops'), an ingredient of beer. Human exposure to xanthohumol and related prenylflavonoids, such as 8-prenylnaringenin and isoxanthohumol, is primarily through beer consumption. Xanthohumol has been characterized a 'broad-spectrum' cancer chemopreventive agent in in vitro studies, while 8-prenylnaringenin enjoys fame as the most potent phytoestrogen known to date. These biological activities suggest that prenylflavonoids from hops have potential for application in cancer prevention programs and in prevention or treatment of (post-)menopausal 'hot flashes' and osteoporosis. Xanthohumol and 8-prenylnaringenin are metabolized into many flavonoid derivatives with modified 3,3-dimethyl allyl (prenyl) moieties. Xanthohumol is formed in lupulin glands by a specialized branch of flavonoid biosynthesis that involves prenylation and O-methylation of the polyketide intermediate chalconaringenin. Although a lupulin gland-specific chalcone synthase is known, the aromatic prenyltransferase and O-methyltransferase participating in xanthohumol have not been identified. The prenylflavonoid pathway is a possible target for breeding or biotechnological modification of hops with the aim of increasing xanthohumol levels for beer brewing and 8-prenylnaringenin levels for pharmaceutical production.

Anticarcinogenic Agents↗

Model for using hip-hop music for small group HIV/AIDS prevention counseling with African American adolescents and young adults.

Currently little attention has been directed, with the exception of peer education efforts, to constructively develop new and innovative ways to promote HIV/AIDS primary prevention among African American (AA) adolescents and young adults. With this in mind, the aim of this conceptual effort is to present a HIV/AIDS preventive counseling protocol developed for use with AA young adults that makes use of hip-hop music, a form of music popularized by young AAs. The author contend that an increased understanding of the relationships that many AA young adults have with hip-hop music may be used by disease prevention personnel to educate these populations about protective factors for HIV. Making use of hip-hop music is one strategy for integrating counseling in prevention and health maintenance. The overall implications of using hip-hop music in health promotion are unlimited. First, this method makes use of cultural relevant materials to address the educational and health needs of the target community. Second, it is grounded in an approach that serves to stimulate cooperative learning based on peer developed content. Moreover, the use of this medium can be applied to other health promotion activities such as violence/harm reduction and substance abuse prevention, upon reviews of songs for appropriate content. The authors contend that such an approach holds heuristic value in dealing with HIV/AIDS prevention among AA young adults. Additional testing of the intervention is warranted in the refinement of this innovative intervention.

Acquired Immunodeficiency Syndrome↗

Charge transport in DNA via thermally induced hopping.

In this contribution we advance and explore the thermally induced hopping (TIH) mechanism for long-range charge transport (CT) in DNA and in large-scale chemical systems. TIH occurs in donor-bridge-acceptor systems, which are characterized by off-resonance donor-bridge interactions (energy gap DeltaE > 0), involving thermally activated donor-bridge charge injection followed by intrabridge charge hopping. We observe a "transition" from superexchange to TIH with increasing the bridge length (i.e., the number N of the bridge constituents), which is manifested by crossing from the exponential N-dependent donor-acceptor CT rate at low N (< N(X)) to a weakly (algebraic) N-dependent CT rate at high N (>N(X)). The "critical" bridge size N(X) is determined by the energy gap, the nearest-neighbor electronic couplings, and the temperature. Experimental evidence for the TIH mechanism was inferred from our analysis of the chemical yields for the distal/proximal guanine (G) triplets in the (GGG)(+)TTXTT(GGG) duplex (X = G, azadine (zA), and adenine (A)) studied by Nakatani, Dohno and Saito [J. Am. Chem. Soc. 2000, 122, 5893]. The TIH sequential model, which involves hole hopping between (GGG) and X, is analyzed in terms of a sequential process in conjunction with parallel reactions of (GGG)(+) with water, and provides a scale of (free) energy gaps (relative to (GGG)(+)) of Delta = 0.21-0.24 eV for X = A, Delta = 0.10-0.14 eV for X = zA, and Delta = 0.05-0.10 eV for X = G. We further investigated the chemical yields for long-range TIH in (G)l(+)Xn(G)l (l = 1-3) duplexes, establishing the energetic constraints (i.e., the donor - bridge base (X) energy gap Delta), the bridge structural constraints (i.e., the intrabridge X-X hopping rates k(m)), and the kinetic constraints (i.e., the rate k(d) for the reaction of with water). Effective TIH is expected to prevail for Delta less than or approximately equal to 0.20 eV with a "fast" water reaction (k(d)/k(m) approximately 10(-3)) and for Delta < 0.30 eV with a "slow" water reaction (k(d)/k(m) approximately 10(-5)). We conclude that (T)n bridges (for which Delta approximately equals 0.6 eV) cannot act in TIH of holes. From an analysis based on the energetics of the electronic coupling matrix elements in G(+)(T-A)n(GGG) duplexes we conclude that the superexchange mechanism is expected to dominate for n = 1-4. For long (A)n bridges (n > or approximately equal to 4) the TIH prevails, provided that the water side reaction is slow, raising the issue of chemical control of TIH through long (A)n bridges in DNA attained by changing the solution composition.

DNA↗

Charge separation in DNA via consecutive adenine hopping.

Charge transfer in DNA is of current interest because of the involvement of charge transfer in oxidative DNA damage and electronic molecular devices. We have investigated the charge separation process via the consecutive adenine (A)-hopping mechanism using laser flash photolysis of DNA conjugated with naphthaldiimide (NDI) as an electron acceptor and phenothiazine (PTZ) as a donor. Upon the 355-nm laser flash excitation of NDI, the charge separation and recombination process between NDI and PTZ was observed. The yields of the charge separation via the consecutive A-hopping were slightly dependent upon the number of A bases between the two chromophores, while the charge recombination rate was strongly dependent upon the distance. The charge-separated state persisted over 300 micros when NDI was separated from PTZ by eight A bases. Furthermore, the rate constant of the A-hopping process was determined to be 2 x 10(10) s(-1) from an analysis of the yield of the charge separation depending on the number of A-hopping steps.

Adenine↗

DNA charge transport: conformationally gated hopping through stacked domains.

The role of base motions in delocalization and propagation of charge through double helical DNA must be established experimentally and incorporated into mechanistic descriptions of DNA-mediated charge transport (CT). Here, we address these fundamental issues by examining the temperature dependence of the yield of CT between photoexcited 2-aminopurine (Ap) and G through DNA bridges of varied length and sequence. DNA assemblies (35-mers) were constructed containing adenine bridges Ap(A)(n)()G (n = 0-9, 3.4-34 A) and mixed bridges, ApAAIAG and ApATATG. CT was monitored through fluorescence quenching of Ap by G and through HPLC analysis of photolyzed DNA assemblies containing Ap and the modified guanine, N(2)-cyclopropylguanosine ((CP)G); upon oxidation, the (CP)G radical cation undergoes rapid ring opening. First, we find that below the duplex melting temperature ( approximately 60 degrees C), the yield of CT through duplex DNA increases with increasing temperature governed by the length and sequence of the DNA bridge. Second, the distance dependence of CT is regulated by temperature; enhanced DNA base fluctuations within duplex DNA extend CT to significantly longer distances, here up to 34 A in <10 ns. Third, at all temperatures the yield of CT does not exhibit a simple distance dependence; an oscillatory component, with a period of approximately 4-5 base pairs, is evident. These data cannot be rationalized by superexchange, hopping of a localized charge injected into the DNA bridge, a temperature-induced transition from superexchange to thermally induced hopping, or by phonon-assisted polaron hopping. Instead, we propose that CT occurs within DNA assemblies possessing specific, well-coupled conformations of the DNA bases, CT-active domains, accessed through base motion. CT through DNA is described as conformationally gated hopping among stacked domains. Enhanced DNA base motions lead to longer range CT with a complex distance dependence that reflects the roles of coherent dynamics and charge delocalization through transient domains. Consequently, DNA CT is not a simple function of distance but is intimately related to the dynamical structure of the DNA bridge.

2-Aminopurine↗

Identification and in vitro biological activities of hop proanthocyanidins: inhibition of nNOS activity and scavenging of reactive nitrogen species.

Oligomeric proanthocyanidins constitute a group of water-soluble polyphenolic tannins that are present in the female inflorescences (up to 5% dry wt) of the hop plant (Humulus lupulus). Humans are exposed to hop proanthocyanidins through consumption of beer. Proanthocyanidins from hops were characterized for their chemical structure and their in vitro biological activities. Chemically, they consist mainly of oligomeric catechins ranging from dimers to octamers, with minor amounts of catechin oligomers containing one or two gallocatechin units. The chemical structures of four procyanidin dimers (B1, B2, B3, and B4) and one trimer, epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin (TR), were elucidated using mass spectrometry, NMR spectroscopy, and chemical degradation. When tested as a mixture, the hop oligomeric proanthocyanidins (PC) were found to be potent inhibitors of neuronal nitric oxide synthase (nNOS) activity. Among the oligomers tested, procyanidin B2 was most inhibitory against nNOS activity. Procyanidin B3, catechin, and epicatechin were noninhibitory against nNOS activity. PC and the individual oligomers were all strong inhibitors of 3-morpholinosydnonimine (SIN-1)-induced oxidation of LDL, with procyanidin B3 showing the highest antioxidant activity at 0.1 microg/mL. The catechin trimer (TR) exhibited antioxidant activity more than 1 order of magnitude greater than that of alpha-tocopherol or ascorbic acid on a molar basis.

Anthocyanins↗

Structural identification and distribution of proanthocyanidins in 13 different hops.

Ten newly isolated hop proanthocyanidin oligomers and flavan-3-ol monomers from 13 different hops have been identified as gallocatechin, gallocatechin-(4alpha-->8)-catechin, gallocatechin-(4alpha-->6)-catechin, catechin-(4alpha-->8)-gallocatechin, catechin-(4alpha-->6)-gallocatechin, afzelechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-catechin-(4alpha-->8)-catechin, epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin, catechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, and gallocatechin-(4alpha-->8)-gallocatechin-(4alpha-->8)-catechin, together with seven previously isolated oligomers, namely, catechin, epicatechin, epicatechin-(4beta-->8)-catechin, epicatechin-(4beta-->8)-epicatechin, catechin-(4alpha-->8)-catechin, catechin-(4alpha-->8)-epicatechin, and epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin. These compounds were subjected to acid-catalyzed degradation in the presence of phloroglucinol or by partial or complete acid-catalyzed degradation and reaction with benzyl mercaptan followed by desulfurization. The resultant adducts when compared to authentic samples by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry served to identify the precursors. The composition of proanthocyanidins from 13 different hops was similar, but the concentration of individual compounds showed some differences, which indicated that hop proanthocyanidin profiles are affected by geographic origin and are variable depending on the cultivars.

Chromatography↗

Fate of xanthohumol and related prenylflavonoids from hops to beer.

The fate of three prenylated flavonoids of the chalcone type, xanthohumol, desmethylxanthohumol, and 3'-geranylchalconaringenin, was monitored with LC/MS-MS from hops (Humulus lupulus L.) to beer in two brewing trials. The three prenylchalcones were largely converted into their isomeric flavanones, isoxanthohumol, prenylnaringenins, and geranylnaringenins, respectively, in the boiling wort. Losses of prenylflavonoids were due to incomplete extraction from the hops into the wort (13-25%), adsorption to insoluble malt proteins (18-26%), and adsorption to yeast cells (11-32%) during fermentation. The overall yield of xanthohumol, after lagering of the beer and largely in the form of isoxanthohumol, amounted to 22-30% of the hops' xanthohumol. About 10% of the hops' desmethylxanthohumol, completely converted into prenylnaringenins, remained in the beers. 3'-Geranylchalconaringenin behaved similarly to desmethylxanthohumol. Solubility experiments indicated that (1) malt carbohydrates form soluble complexes with xanthohumol and isoxanthohumol and (2) solubility does not dictate the isoxanthohumol levels of finished beers.

Beer↗

Field-testing of methyl salicylate for recruitment and retention of beneficial insects in grapes and hops.

Evidence for recruitment and retention of beneficial insects in grapes and hops using controlled-release dispensers of methyl salicylate (MeSA), a component of herbivore-induced volatile blends, is presented. In a replicated experiment conducted in a juice grape vineyard, sticky cards in blocks baited with MeSA captured significantly greater numbers of five species of predatory insects (Chrysopa nigricornis, Hemerobius sp., Deraeocoris brevis, Stethorus punctum picipes, Orius tristicolor) than unbaited blocks. Four insect families (Syrphidae, Braconidae, Empididae, Sarcophagidae) were also significantly more abundant in the MeSA-baited blocks, as indicated by sticky card captures. Canopy shake samples and sticky card monitoring conducted in a MeSA-baited, unsprayed hop yard indicated development and maintenance of a beneficial arthropod population that was nearly four times greater than that present in an unbaited reference yard. Four times as many S. punctum picipes and six times as many O. tristicolor were sampled in the MeSA yard. Similar contrasts in abundance of these predators and others were apparent when compared with levels recorded in the yard in previous years. The large population of predatory insects in the MeSA-baited hop yard was associated with a dramatic reduction in spider mite numbers, the major arthropod pest of hops, in late June, and subeconomic populations were maintained for the rest of the season. The evidence presented here is highly suggestive that the use of controlled-release MeSA in a crop could increase recruitment and residency of populations of certain beneficial insects. This strategy may have the potential to enhance the efficacy and reliability of conservation biological control in crop pest management.

Animals↗

Individual factors affecting stabilometry and one-leg hop test in 75 healthy subjects, aged 15-44 years.

The aims of this study were: 1) to investigate whether single-limb stabilometry and a one-leg hop test are influenced by age, sex, height, weight or activity level in healthy subjects, 2) to examine possible differences between the right and the left leg, 3) to determine Limb Symmetry Index (LSI) values, and 4) to study the relation between the stabilometric variables, that is, average speed (AS) and amplitude of center of pressure (CP) movements. Seventy-five healthy subjects (39 women) were studied, with a mean age of 29.5 years (SD 8.2, range 15-44), mean height 175 cm (SD 8.6, range 155-194), mean weight 67.8 kg (SD 9.7, range 50-90) and median value 8 (quartiles 8-9, range 7-10) on a 1-10 activity-level scale. AS was higher among the men compared to the women and increased with increasing age. The females hopped shorter distances than the males. The hop distance decreased with increasing age among the women. Height, weight and activity level within the actual range had no significant influence on stabilometric or hop-test values. When comparing patients with healthy subjects, they should be matched according to sex and age, with an age difference of less than 10 years within pairs.

Adolescent↗

Direct observation of hole transfer through DNA by hopping between adenine bases and by tunnelling.

The function of DNA during oxidative stress and its suitability as a potential building block for molecular devices depend on long-distance transfer of electrons and holes through the molecule, yet many conflicting measurements of the efficiency of this process have been reported. It is accepted that charges are transported over long distances through a multistep hopping reaction; this 'G-hopping' involves positive charges moving between guanines (Gs), the DNA bases with the lowest ionization potential. But the mechanism fails to explain the persistence of efficient charge transfer when the guanine sites are distant, where transfer rates do not, as expected, decrease rapidly with transfer distance. Here we show experimentally that the rate of charge transfer between two guanine bases decreases with increasing separation only if the guanines are separated by no more than three base pairs; if more bridging base pairs are present, the transfer rates exhibit only a weak distance dependence. We attribute this distinct change in the distance dependence of the rate of charge transfer through DNA to a shift from coherent superexchange charge transfer (tunnelling) at short distances to a process mediated by thermally induced hopping of charges between adenine bases (A-hopping) at long distances. Our results confirm theoretical predictions of this behaviour, emphasizing that seemingly contradictory observations of a strong as well as a weak influence of distance on DNA charge transfer are readily explained by a change in the transfer mechanism.

Adenine↗

Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p.

Coupling of Rab GTPase activation and SNARE complex assembly during membrane fusion is poorly understood. The homotypic fusion and vacuole protein sorting (HOPS) complex links these two processes: it is an effector for the vacuolar Rab GTPase Ypt7p and is required for vacuolar SNARE complex assembly. We now report that pure, active HOPS complex binds phosphoinositides and the PX domain of the vacuolar SNARE protein Vam7p. These binding interactions support HOPS complex association with the vacuole and explain its enrichment at the same microdomains on docked vacuoles as phosphoinositides, Ypt7p, Vam7p, and the other SNARE proteins. Concentration of the HOPS complex at these microdomains may be a key factor for coupling Rab GTPase activation to SNARE complex assembly.

Membrane Fusion↗

Aha1 competes with Hop, p50 and p23 for binding to the molecular chaperone Hsp90 and contributes to kinase and hormone receptor activation.

The ATP-dependent molecular chaperone Hsp90 (heat-shock protein 90) is essential for the maturation of hormone receptors and protein kinases. During the process of client protein activation, Hsp90 co-operates with cofactors/co-chaperones of unique sequence, e.g. Aha1 (activator of Hsp90 ATPase 1), p23 or p50, and with cofactors containing TPR (tetratricopeptide repeat) domains, e.g. Hop, immunophilins or cyclophilins. Although the binding sites for these different types of cofactors are distributed along the three domains of Hsp90, sterical overlap and competition for binding sites restrict the combinations of cofactors that can bind to Hsp90 at the same time. The recently discovered cofactor Aha1 associates with the middle domain of Hsp90, but its relationship to other cofactors of the molecular chaperone is poorly understood. Therefore we analysed whether complexes of Aha1, p23, p50, Hop and a cyclophilin with Hsp90 are disrupted by the other four cofactors by gel permeation chromatography using purified proteins. It turned out that Aha1 competes with the early cofactors Hop and p50, but can bind to Hsp90 in the presence of cyclophilins, suggesting that Aha1 acts as a late cofactor of Hsp90. In contrast with p50, which can bind to Hop, Aha1 does not interact directly with any of the other four cofactors. In vivo studies in yeast and in mammalian cells revealed that Aha1 is not specific for kinase activation, but also contributes to maturation of hormone receptors, proposing a general role for this cofactor in the activation of Hsp90-dependent client proteins.

Binding, Competitive↗