The Photosensitive Retinal Pigment System of Gekko gekko

Retinal extracts of Gekko gekko were found to contain two retinene₁ photopigments, one with maximum absorption at about 521 mµ, the second with a maximum in the region of 478 mµ. These pigments were assayed by the method of partial bleaching and their spectral characteristics studied by examining their difference spectra. The 478 mµ pigment was present in the extracts as 8 per cent of the total photopigment concentration. The two pigment systems were shown to be biochemically independent and to have different properties. Unlike the 521 mµ pigment, for example, the 478 mµ pigment was found to resist the action of NH₂OH and, within the cells, to be unaffected by sucrose solutions. These solutions destroyed or altered the 521 system so that extracts of sucrose-treated retinae were found to contain significantly less 521 photopigment. In digitonin solution the 521 pigment was unaffected by sucrose treatment. Both pigments were extracted from separated, washed outer segments and so are considered to be visual pigments. This dual system accounts for the spectral sensitivity of this gecko as determined by Denton. A search was made, but no evidence was secured for the presence of a photopigment absorbing at longer wavelengths. Electoretinographic data suggest, however, that an elevated sensitivity at longer wavelengths occurs in some geckos so that a continued search is justified for a third photopigment.     

紫细菌光合色素指纹图谱的建立与色素分析

【目的】探索快速高效的色素样品制备方法;为建立紫细菌全色素TLC和HPLC标准指纹图谱和数据库提供研究方法和思路;为色素代谢与调控等研究提供快速的色素分析方法。【方法】选择沼泽红假单胞菌(Rhodopesudomonas palustris CQV97)为材料,采用改良丙酮甲醇提取法、TLC重复展层法、图像灰度分析法、吸收光谱法、HPLC和MS法进行色素分析。【结果】甲醇或丙酮可选择性地提取样品的细菌叶绿素和类胡萝卜素,通过对丙酮甲醇法的改良,使色素提取总量提高了13.5%。建立了CQV97菌株全色素的TLC和HPLC指纹图谱,二者均含有11种色素组分。图像灰度分析法估算了TLC指纹图谱各色素组分的表观相对含量。以TLC图谱的各色素组分为外标物,明确了HPLC图谱中各色素组分的保留时间(Rt)与TLC图谱中各色素组分的迁移率(Rf)之间的对应关系。结合色素代谢途径、光谱分析和MS,定性分析了指纹图谱中11种色素组分。TLC或HPLC分析结果表明,理论样品与对照样品色素组分和含量一致,而实际样品与对照样品色素组分一致,但含量不同,重复测定3次,样品中色素相对含量的RSD均小于5%。【结论】改良的丙酮甲醇法可以快速高效地提取光合细菌的色素。采用TLC重复展层法和HPLC法建立的全色素指纹图谱色素组成一致,重复稳定性好,各具特色。TLC和HPLC两种指纹图谱分析方法均能进行光合色素的快速分析,适合于紫细菌色素合成途径中主要积累色素的组分和含量变化规律研究。     

Visual Pigments of Goldfish Cones : Spectral Properties and Dichroism

Freshly isolated retinal photoreceptors of goldfish were studied microspectrophotometrically. Absolute absorptance spectra obtained from dark-adapted cone outer segments reaffirm the existence of three spectrally distinct cone types with absorption maxima at 455 ± 3,530 ± 3, and 625 ± 5 nm. These types were found often recognizable by gross cellular morphology. Side-illuminated cone outer segments were dichroic. The measured dichroic ratio for the main absorption band of each type was 2–3:1. Rapidly bleached cells revealed spectral and dichroic transitions in regions near 400–410, 435–455, and 350–360 nm. These photoproducts decay about fivefold as fast as the intermediates in frog rods. The spectral maxima of photoproducts, combined with other evidence, indicate that retinene₂ is the chromophore of all three cone pigments. The average specific optical density for goldfish cone outer segments was found to be 0.0124 ± 0.0015/µm. The spectra of the blue-, and green-absorbing cones appeared to match porphyropsin standards with half-band width Δν = 4,832 ± 100 cm^–1. The red-absorbing spectrum was found narrower, having Δν = 3,625 ± 100 cm^–1. The results are consistent with the notion that visual pigment concentration within the outer segments is about the same for frog rods and goldfish cones, but that the blue-, and green-absorbing pigments possess molar extinctions of 30,000 liter/mol cm. The red-absorbing pigment was found to have extinction of 40,000 liter/mol cm, assuming invariance of oscillator strength among the three cone spectra.     

Cone photopigments in nocturnal and diurnal procyonids

Summary Procyonids are small, New World carnivores distributed among some 6 genera. Electroretinogram (ERG) flicker photometry was used to measure the spectra of the cone photopigments for members of two nocturnal species, the raccoon (Procyon lotor) and the kinkajou (Potos flavus), and a diurnal species, the coati (Nasua nasua). Each of the 3 has a class of cone photopigment with maximum sensitivity in the middle to long wavelengths. The spectral positioning of this cone is different for the three. Whereas the raccoon and kinkajou are monochromatic, the diurnal coati is a dichromat having an additional class of cone photopigment with peak sensitivity close to 433 nm.Abbreviations ERG electroretinogram - SWS short wavelength sensitive     

Absorption and Screening in Phycomyces

In vivo absorption measurements were made through the photosensitive zones of Phycomyces sporangiophores and absorption spectra are presented for various growth media and for wavelengths between 400 and 580 mµ. As in mycelia, β-carotene was the major pigment ordinarily found. The addition of diphenylamine to the growth media caused a decrease in β-carotene and an increase in certain other carotenoids. Growth in the dark substantially reduced the amount of β-carotene in the photosensitive zone; however, growth on a lactate medium failed to suppress β-carotene in the growing zone although the mycelia appeared almost colorless. Also when diphenylamine was added to the medium the absorption in the growing zone at 460 mµ was not diminished although the colored carotenoids in the bulk of the sporangiophore were drastically reduced. Absorption which is characteristic of the action spectra was not found. Sporangiophores immersed in fluids with a critical refractive index show neither positive nor negative tropism. Measurements were made of the critical refractive indices for light at 495 and 510 mµ. The critical indices differed only slightly. Assuming primary photoreceptors at the cell wall, the change in screening due to absorption appears too large to be counterbalanced solely by a simple effect of the focusing change. The possibility is therefore advanced that the receptors are internal to most of the cytoplasm; i.e., near the vacuole.     

Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation

Background

Platelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization.

Methodology/Principal Findings

In order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels.

Significance/Conclusion

Our data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells.     

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.     

Opsins and mammalian photoentrainment

Research over the past decade has provided overwhelming evidence that photoreception in the vertebrate eye is not confined to the rod and cone photoreceptors. It appears that photoreceptor cells within the inner retina provide irradiance information to a wide variety of different photosensory tasks including photoentrainment, pupillary constriction and masking behaviour. Action spectra in mice lacking all rod and cone photoreceptors ( rd/rd cl) have demonstrated the existence of a previously uncharacterised, opsin/vitamin-A-based photopigment with peak sensitivity at 479 nm (opsin photopigment/OP(479)). The review addresses the question: has the gene encoding OP(479) already been isolated, and if not, what type of gene should we be seeking and where in the eye might this gene be expressed? On the basis of available data, the gene that encodes OP(479) remains unidentified, and two broad possibilities exist. On the assumption that OP(479) will be like all of the other vertebrate photopigments (ocular and extraocular) and share a close phylogenetic relationship based upon amino acid identity and a conserved genomic structure, then the gene encoding OP(479) has yet to be isolated. Alternatively, there may have been a separate line of photopigment evolution in the vertebrates that has given rise to the melanopsin family. If true then the mammalian melanopsin gene may encode OP(479). Only when melanopsin and other candidates for OP(479) have been functionally expressed, and shown to encode a photopigment that matches the action spectrum of OP(479), can firm conclusions about the identity of the non-rod, non-cone ocular photoreceptor of mammals be made.     

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.     

Beta-Catenin and Plakoglobin Expression during Zebrafish Tooth Development and Replacement

We analyzed the protein distribution of two cadherin-associated molecules, plakoglobin and β-catenin, during the different stages of tooth development and tooth replacement in zebrafish. Plakoglobin was detected at the plasma membrane already at the onset of tooth development in the epithelial cells of the tooth. This pattern remained unaltered during further tooth development. The mesenchymal cells only showed plakoglobin from cytodifferentiation onwards. Plakoglobin 1a morpholino-injected embryos showed normal tooth development with proper initiation and differentiation. Although plakoglobin is clearly present during normal odontogenesis, the loss of plakoglobin 1a does not influence tooth development. β-catenin was found at the cell borders of all cells of the successional lamina but also in the nuclei of surrounding mesenchymal cells. Only membranous, not nuclear, β-catenin, was found during morphogenesis stage. However, during cytodifferentiation stage, both nuclear and membrane-bound β-catenin was detected in the layers of the enamel organ as well as in the differentiating odontoblasts. Nuclear β-catenin is an indication of an activated Wnt pathway, therefore suggesting a possible role for Wnt signalling during zebrafish tooth development and replacement.     

High Cholesterol Diet Induces IL-1β Expression in Adult but Not Larval Zebrafish

Recently, it has been demonstrated that high cholesterol diet induced hypercholesterolemia and vascular lipid oxidation and accumulation in zebrafish larvae, suggesting that zebrafish is a new promising atherosclerosis model in addition to mouse models. However, up to date, there was no report regarding inflammatory cytokine expression during the lipid accumulation in zebrafish larva and adult fish. In this study, we first demonstrated the expression levels of IL-1β and TNF-α in high cholesterol diet (HCD)-fed zebrafish larvae, and found that although HCD induced vascular lipid accumulation, the cytokine expressions in the larvae were not changed by HCD. Furthermore, there was no significant difference in leukocyte accumulation in vessels between control and HCD fed group. But prolonged HCD induced IL-1β expression in spleen and liver compared to those of control zebrafish, and produced very early stage of fatty streak lesion in dorsal aorta of 19 week HCD-fed zebrafish. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis.     

A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish

Inhibition of amyloid β-peptide (Aβ) production by blocking γ-secretase activity is at present one of the most promising therapeutic strategies to slow progression of Alzheimer’s disease pathology. γ-secretase inhibitors apparently block Aβ generation via interference with presenilin (PS) function. Besides being an essential component of the γ-secretase complex, PS itself may be an aspartyl protease with γ-secretase activity, which is not only required for Aβ production but also for a similar proteolytic process involved in Notch signaling. Here we demonstrate that treatment of zebrafish embryos with a known γ-secretase inhibitor affects embryonic development in a manner indistinguishable from Notch signaling deficiencies at morphological, molecular and biochemical levels. This indicates severe side-effects of γ-secretase inhibitors in any Notch-dependent cell fate decision and demonstrates that the zebrafish is an ideal vertebrate system to validate compounds that selectively affect Aβ production, but not Notch signaling, under in vivo conditions.     

ENaC proteolytic regulation by channel-activating protease 2

Epithelial sodium channels (ENaCs) perform diverse physiological roles by mediating Na⁺ absorption across epithelial surfaces throughout the body. Excessive Na⁺ absorption in kidney and colon elevates blood pressure and in the airways disrupts mucociliary clearance. Potential therapies for disorders of Na⁺ absorption require better understanding of ENaC regulation. Recent work has established partial and selective proteolysis of ENaCs as an important means of channel activation. In particular, channel-activating transmembrane serine proteases (CAPs) and cognate inhibitors may be important in tissue-specific regulation of ENaCs. Although CAP2 (TMPRSS4) requires catalytic activity to activate ENaCs, there is not yet evidence of ENaC fragments produced by this serine protease and/or identification of the site(s) where CAP2 cleaves ENaCs. Here, we report that CAP2 cleaves at multiple sites in all three ENaC subunits, including cleavage at a conserved basic residue located in the vicinity of the degenerin site (α-K561, β-R503, and γ-R515). Sites in α-ENaC at K149/R164/K169/R177 and furin-consensus sites in α-ENaC (R205/R231) and γ-ENaC (R138) are responsible for ENaC fragments observed in oocytes coexpressing CAP2. However, the only one of these demonstrated cleavage events that is relevant for the channel activation by CAP2 takes place in γ-ENaC at position R138, the previously identified furin-consensus cleavage site. Replacement of arginine by alanine or glutamine (α,β,γR138A/Q) completely abolished both the Na⁺ current (I_Na) and a 75-kD γ-ENaC fragment at the cell surface stimulated by CAP2. Replacement of γ-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced I_Na and the 75-kD γ-ENaC fragment. These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in γ-ENaC.     

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.     

Effect of Light Quality on Phycobilisome Components of the Cyanobacterium Spirulina platensis

Phycobilisomes from the nonchromatic adapting cyanobacterium Spirulina platensis are composed of a central core containing allophycocyanin and rods with phycocyanin and linker polypeptides in a regular array. Room temperature absorption spectra of phycobilisomes from this organism indicated the presence of phycocyanin and allophycocyanin. However, low temperature absorption spectra showed the association of a phycobiliviolin type of chromophore within phycobilisomes. This chromophore had an absorption maximum at 590 nanometers when phycobilisomes were suspended in 0.75 molar K-phosphate buffer (pH 7.0). Purified phycocyanin from this cyanobacterium was found to consist of three subparticles and the phycobiliviolin type of chromophore was associated with the lowest density subparticle. Circular dichroism spectra of phycocyanin subparticles also indicated the association of this chromophore with the lowest density subparticle. Absorption spectral analysis of α and β subunits of phycocyanin showed that phycobiliviolin type of chromophore was attached to the α subunit, but not the β subunit. Effect of light quality showed that green light enhanced the synthesis of this chromophore as analyzed from the room temperature absorption spectra of phycocyanin subparticles and subunits, while red or white light did not have any effect. Low temperature absorption spectra of phycobilisomes isolated from green, red, and white light conditions also indicated the enhancement of phycobiliviolin type of chromophore under green light.     

Structural Insights into the Neutralization Properties of the Fully Human,Anti-interferon Monoclonal Antibody Sifalimumab

We report the three-dimensional structure of human interferon α-2A (IFN-α2A) bound to the Fab fragment of a therapeutic monoclonal antibody (sifalimumab; IgG1/κ). The structure of the corresponding complex was solved at a resolution of 3.0 Å using molecular replacement and constitutes the first reported structure of a human type I IFN bound to a therapeutic antibody. This study revealed the major contribution made by the first complementarity-determining region in each of sifalimumab light and heavy chains. These data also provided the molecular basis for sifalimumab mechanism of action. We propose that its interferon-neutralizing properties are the result of direct competition for IFN-α2A binding to the IFN receptor subunit 1 (IFNAR1) and do not involve inhibiting IFN-α2A binding to the IFN receptor subunit 2 (IFNAR2).     

Fluorescent Protein Voltage Probes Derived from ArcLight that Respond to Membrane Voltage Changes with Fast Kinetics

We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (τ1-on ~10 ms, τ2-on ~ 50 ms) are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (τ) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the Ciona-based ArcLight (~35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals.     

The epithelial sodium channel in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Epithelial sodium channel (ENaC) is a Na⁺-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na⁺ absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, β and γ proteins. The nENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.     

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

Retinal Cone Photoreceptors Require Phosducin-Like Protein 1 for G Protein Complex Assembly and Signaling

G protein β subunits (Gβ) play essential roles in phototransduction as part of G protein βγ (Gβγ) and regulator of G protein signaling 9 (RGS9)-Gβ₅ heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (G_t2) and RGS9-Gβ₅ subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ₅ assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling.