"Immunolocalization and effect of low concentrations of Insulin like growth factor-1 (IGF-1) in the canine ovary".

Insulin like growth factor-1 (IGF-1) plays an important role in the regulation of ovarian function. Despite its extensive study in several species, there is a paucity of information about IGF-1`s function and localization in the canine ovary. The aim of the present study was to assess the effect of IGF-1 on oocyte nuclear maturation and to immunolocalize the IGF-1 and its receptor (IGF-1R) in the ovary. Cumulus-oocyte complexes (COCs) were obtained from 34 bitches. The COCs from each bitch were incubated in TCM 199-HEPES in the absence (n = 199) or presence (n = 204) of 100 ng/ml IGF-1 for 96 hr at 38ºC in 5% CO2 , stained and evaluated for nuclear maturation by fluorescence microscopy. The results showed that the addition of IGF-1 did not have an effect (p Ëƒ 0.05) on the nuclear maturation under these conditions. The immunohistochemical study revealed nuclear and cytoplasmic staining for IGF-1 and IGF-1R, respectively. Both were localized in all ovarian structures including the corpus luteum, but not in the granulosa cells from primordial follicles. In addition, IGF-1 was not localized in the oocytes in tertiary follicles. The results obtained show the presence of IGF-1 through the stages of follicular growth and in the corpus luteum of the canine ovary. However, its role on oocyte nuclear maturation could not be demonstrated.

Vitrification of cat ovarian tissue: Does fragment size matters?

The aim of the present study was to investigate whether or not the size of the ovarian fragment influences its resistance to cryostorage. For that purpose, ovaries were collected from 34 queens (various breeds, age 1-5 year) by routine ovariectomy, transported to the laboratory and then sectioned in different sizes (3 mm × 3 mm × 3 mm, 5 mm × 3 mm × 3 mm and 7 mm × 3 mm × 3 mm) and randomly assigned to a control (GC3, GC5 and GC7, respectively) or vitrified (GV3, GV5 and GV7, respectively) groups. Vitrified-warmed fragments were evaluated by histomorphology and immunohistochemistry (for apoptotic rates by using cleaved caspase-3). Histological examination reveals that 72.97% of the follicles in GV3 and 72.58% in GV5 were normal while only 42.86% of the follicles in GV7. The main morphological alteration presented in all groups was a detachment of the epithelial cells. Similarly, immunohistochemistry evaluation using caspase 3 revealed a small proportion of apoptotic cells in GV3 (8.43%) while in GV7 30.43% of the cells expressed cleaved caspase-3. These findings indicate that fragments sectioned in 3 mm × 3 mm × 3 mm (27 mm3 ) seem more adequate for perfusion of the cryoprotectant, causing less damage to the cell after vitrification-warming.

Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra.

Escherichia coli is the most common bacterial agent isolated from canine pyometra. The frequencies of 24 virulence genes and pulsed field gel electrophoresis (PFGE) profiles were determined for 23 E. coli isolates from cases of canine pyometra in Brazil. The frequencies of virulence genes were 91.3% fimH, 91.3% irp-2, 82.6% fyuA, 56.5% iroN, 47.8% traT, 39.1% usp, 34.8% sfaD/E, 34.8% tsh, 30.4% papC, 30.4% hlyA, 26.1% papGIII, 26.1% cnf-1, 21.7% papE/F, 21.7% iss, 17.4% iutA, 17.4% ompT, 17.4% cvaC, 17.4% hlyF, 17.4% iucD, 13.0% iucC, 13.0% astA, 4.3% papGII, 0% afaB/C and 0% papGI. The high frequency of yersiniabactin (fyuA and irp2) and salmochelin (iroN) genes suggests that iron uptake systems might be important in the pathogenesis of canine pyometra. PFGE profiles of 19 isolates were heterogeneous, confirming that E. coli isolates from canine pyometra are unlikely to be epidemic clones.

Risk factors for depressive symptoms in a large population on chronic hemodialysis.

Despite their significant influence on the quality of life, depressive symptoms are not usually included as a clinical parameter in the evaluation of hemodialysis patients. We aimed to identify depressive symptoms and associated risk factors in a large group of individuals with end-stage renal disease (ESRD) on chronic hemodialysis. This was a cross-sectional study of 400 consecutive patients. Cases were analyzed according to the presence/absence of depressive symptoms. All individuals were investigated by interview, and all variables were measured concurrently. Depressive symptoms were evaluated by the Beck Depression Inventory (BDI-II ≥16) and sleep quality by the Pittsburgh Sleep Quality Index (PSQI > 5). Among the 400 patients (59% male), depressive symptoms were present in 77 (19.3%). Depressive symptoms were more common in women and were independently associated with poor sleep quality (P = <0.005), unemployment (P = 0.001), diabetes (P = 0.02), hypoalbuminemia (P = 0.01), low education (P = 0.03), and pruritus (P = 0.04). Women with ESRD on chronic hemodialysis are at increased risk of depression. Furthermore, unemployment and the presence of diabetes, hypoalbuminemia, low education, and pruritus are significantly associated with depressive symptoms. Depressive symptoms are also independently associated with poor quality sleep and studies about the effects of sleep hygiene therapy on depressive symptoms are warranted.

The type 1 insulin-like growth factor receptor (IGF-IR) pathway is mandatory for the follistatin-induced skeletal muscle hypertrophy.

Myostatin inhibition by follistatin (FS) offers a new approach for muscle mass enhancement. The aim of the present study was to characterize the mediators responsible for the FS hypertrophic action on skeletal muscle in male mice. Because IGF-I and IGF-II, two crucial skeletal muscle growth factors, are induced by myostatin inhibition, we assessed their role in FS action. First, we tested whether type 1 IGF receptor (IGF-IR) is required for FS-induced hypertrophy. By using mice expressing a dominant-negative IGF-IR in skeletal muscle, we showed that IGF-IR inhibition blunted by 63% fiber hypertrophy caused by FS. Second, we showed that FS caused the same degree of fiber hypertrophy in wild-type and IGF-II knockout mice. We then tested the role of the signaling molecules stimulated by IGF-IR, in particular the Akt/mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (S6K) pathway. We investigated whether Akt phosphorylation is required for the FS action. By cotransfecting a dominant-negative form of Akt together with FS, we showed that Akt inhibition reduced by 65% fiber hypertrophy caused by FS. Second, we evaluated the role of mTOR in FS action. Fiber hypertrophy induced by FS was reduced by 36% in rapamycin-treated mice. Finally, because the activity of S6K is increased by FS, we tested its role in FS action. FS caused the same degree of fiber hypertrophy in wild-type and S6K1/2 knockout mice. In conclusion, the IGF-IR/Akt/mTOR pathway plays a critical role in FS-induced muscle hypertrophy. In contrast, induction of IGF-II expression and S6K activity by FS are not required for the hypertrophic action of FS.

Quality of sleep and day-time sleepiness in chronic hemodialysis: a study of 400 patients.

OBJECTIVE: Impaired sleep has potential health consequences in chronic hemodialysis patients. To date, this issue has not been examined in studies involving a large number of subjects. This study aimed to identify factors associated with poor sleep quality and excessive day-time sleepiness (EDS) in dialysis patients. MATERIAL AND METHODS: This cross-sectional observational study involved 400 patients (59% male) from three hemodialysis centers (SD-HEMOFOR). Quality of sleep was evaluated by the Pittsburgh Sleep Quality Index (PSQI), EDS by the Epworth Sleepiness Scale (ESS), risk of obstructive sleep apnea (OSA) by the Berlin questionnaire and comorbidity severity by the Charlson Comorbidity Index (CCI). RESULTS: Poor sleep quality (PSQI >5) was found in 227 individuals (57%) and was associated with older age (p = 0.001), diabetes (p = 0.03), heart failure (p < 0.005), hypoalbuminemia (p = 0.01), low transferrin saturation (TSAT) (p = 0.009), higher CCI score (p = 0.01) and depression (p < 0.005). Independent factors were older age, heart failure, low TSAT and depressive symptoms. Day-time somnolence was present in 108 patients (27%) and was independently associated with stroke [odds ratio (OR) = 2.84, CI 1.03-7.76), lower hemoglobin concentration (OR = 2.45, CI 0.95-3.03) and high risk of OSA (OR = 1.65, CI 1.03-2.63). High risk of OSA (n = 120; 30%), was associated with hypertension (p < 0.001), overweight/obesity (p = 0.001), older age (p = 0.003) and symptoms of depression (p = 0.01). CONCLUSIONS: Poor sleep quality and EDS were prevalent on chronic hemodialysis. Heart failure, low TSAT and depressive symptoms were independently associated with poor sleep quality. Stroke, anemia and high risk of OSA were independently associated with EDS. These results provide new insight into possible treatment strategies.

Functional assessment of skeletal muscle in intact mice lacking myostatin by concurrent NMR imaging and spectroscopy.

Inhibiting myostatin (mstn) causes spectacular increase in muscle mass, spurring research for therapeutic approaches against neuromuscular disorders. Yet, possible functional deterioration and compromised force production have been reported in isolated muscle of null mstn(-/-) mice. We analyzed vascular and metabolic response to repeated electro-stimulated exercise in vivo in mstn(-/-) mice compared with FVB wild-type controls (WT), using interleaved multi-parametric functional nuclear magnetic resonance (NMR) imaging and spectroscopy. At steady-state exercise, specific force of plantar flexion, phosphocreatine consumption measured by phosphorus spectroscopy and maximum perfusion measured by arterial spin-labeled (ASL) NMR imaging were identical in both groups. After exercise, phosphorus spectroscopy revealed reduced oxidative mitochondrial capacity in mstn(-/-), whereas early recovery perfusion was identical and oxygen extraction, estimated from the blood oxygen level-dependent (BOLD) contrast, was decreased when compared with WT. Hyperemia was prolonged, indicating specific regulation of the perfusional response in mstn(-/-) mice. Histology showed an increased proportion of type IIb fibers in hypertrophied muscles, but the distribution of capillary contacts per fiber between oxidative and glycolytic fibers was unaltered in mstn(-/-) compared with WT. These integrated results formed coherent evidence of a congruous, non-pathologic shift toward a more glycolytic metabolism in this model of mstn(-/-).

Mechanisms of muscle atrophy induced by glucocorticoids.

BACKGROUND: Many pathological states characterized by muscle atrophy (e.g., sepsis, cachexia, starvation, metabolic acidosis and severe insulinopenia) are associated with an increase in circulating glucocorticoid (GC) levels, suggesting that GC could trigger the muscle atrophy observed in these conditions. GC-induced muscle atrophy results from decreased protein synthesis and increased protein degradation. The inhibitory effect of GCs on protein synthesis is thought to result mainly from the inhibition of the p70 ribosomal S6 protein kinase. The stimulatory effect of GCs on muscle proteolysis results from the activation of two major cellular proteolytic systems: ubiquitin proteasome and lysosomal systems. The decrease in muscle production of insulin-like growth factor I (IGF-I), a muscle anabolic growth factor, could contribute to GC-induced muscle atrophy. By activating the phosphatidylinositol-3-kinase/Akt pathway, IGF-I overrides GC action to stunt muscle atrophy. Evidence also indicates that increased production of myostatin, a catabolic growth factor, could play a critical role in GC-induced muscle atrophy. CONCLUSIONS: Recent progress in understanding the role of growth factors in GC-induced muscle atrophy allows investigation into new therapies to minimize this myopathy.

Experimental infection of pregnant queens with two major Brazilian clonal lineages of Toxoplasma gondii.

Toxoplasma gondii isolates from Brazil are biologically and genetically different from European and North America isolates. Recently, four genotypes were considered the common clonal lineages in Brazil and were designated as types BrI, BrII, BrIII, and BrIV. The pathogenicity of two major Brazilian lineages was investigated after oral inoculation of queens in the middle third of their pregnancies with T. gondii cysts. Twelve pregnant queens without T. gondii antibodies were distributed in group A (infected with a type BrI isolate); group 2 (infected with type BrIII isolate), and group 3 (non-infected control). Infection with type BrI isolate caused toxoplasmosis manifestations and abortion from one litter. Toxoplasmosis manifestations besides premature stillbirth of one litter were observed in queens infected with type BrIII isolate. Indirect fluorescence antibody test showed T. gondii antibodies in all eight infected queens at 30 days after inoculation. In two 10-day-old kittens of the same litter (group 1), titers of 16 and 64 were detected. At the same time, titers of 16, 32, and 32 were detected in three kittens from the same litter (group 2). Experimental infection with tissue cysts from a type BrI and type BrIII isolates of T. gondii developed similar reproductive disturbance in primary infected pregnant queens.

Mechanisms of glucocorticoid-induced myopathy.

Glucocorticoid-induced muscle atrophy is characterized by fast-twitch or type II muscle fiber atrophy illustrated by decreased fiber cross-sectional area and reduced myofibrillar protein content. Muscle proteolysis, in particular through the ubiquitin- proteasome system (UPS), is considered to play a major role in the catabolic action of glucocorticoids. The stimulation by glucocorticoids of the UPS is mediated through the increased expression of several atrogenes ('genes involved in atrophy'), such as atrogin-1 and MuRF-1, two ubiquitin ligases involved in the targeting of protein to be degraded by the proteasome machinery. Glucocorticoids also exert an anti-anabolic action by blunting muscle protein synthesis. These changes in protein turnover may result from changes in the production of two growth factors which control muscle mass, namely IGF-I and myostatin respectively anabolic and catabolic toward the skeletal muscle. The decreased production of IGF-I as well as the increased production of myostatin have been both demonstrated to contribute to the muscle atrophy caused by glucocorticoids. At the molecular level, IGF-I antagonizes the catabolic action of glucocorticoids by inhibiting, through the PI3-kinase/Akt pathway, the activity of the transcription factor FOXO, a major switch for the stimulation of several atrogenes. These recent progress in the understanding of the glucocorticoid-induced muscle atrophy should allow to define new therapies aiming to minimize this myopathy. Promising new therapeutic approaches for treating glucocorticoid-induced muscle atrophy are also presented in this review.

Myostatin gene deletion prevents glucocorticoid-induced muscle atrophy.

Glucocorticoids mediate muscle atrophy in many catabolic states. Myostatin expression, a negative regulator of muscle growth, is increased by glucocorticoids and myostatin overexpression is associated with lower muscle mass. This suggests that myostatin is required for the catabolic effects of glucocorticoids. We therefore investigated whether myostatin gene disruption could prevent muscle atrophy caused by glucocorticoids. Male myostatin knockout (KO) and wild-type mice were subjected to dexamethasone treatment (1 mg/kg.d for 10 d or 5 mg/kg.d for 4 d). In wild-type mice, daily administration of low-dose dexamethasone for 10 d resulted in muscle atrophy (tibialis anterior: -15%; gastrocnemius: -13%; P < 0.01) due to 15% decrease in the muscle fiber cross-sectional area (1621 +/- 31 vs. 1918 +/- 64 microm(2), P < 0.01). In KO mice, there was no reduction of muscle mass nor fiber cross-sectional area after dexamethasone treatment. Muscle atrophy after 4 d of high-dose dexamethasone was associated with increased mRNA of enzymes involved in proteolytic pathways (atrogin-1, muscle ring finger 1, and cathepsin L) and increased chymotrypsin-like proteasomal activity. In contrast, the mRNA of these enzymes and the proteasomal activity were not significantly affected by dexamethasone in KO mice. Muscle IGF-I mRNA was paradoxically decreased in KO mice (-35%, P < 0.05); this was associated with a potentially compensatory increase of IGF-II expression in both saline and dexamethasone-treated KO mice (2-fold, P < 0.01). In conclusion, our results show that myostatin deletion prevents muscle atrophy in glucocorticoid-treated mice, by blunting the glucocorticoid-induced enhanced proteolysis, and suggest an important role of myostatin in muscle atrophy caused by glucocorticoids.

Insulin-like growth factor-I gene transfer by electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats.

Catabolic states caused by injury are characterized by a loss of skeletal muscle. The anabolic action of IGF-I on muscle and the reduction of its muscle content in response to injury suggest that restoration of muscle IGF-I content might prevent skeletal muscle loss caused by injury. We investigated whether local overexpression of IGF-I protein by gene transfer could prevent skeletal muscle atrophy induced by glucocorticoids, a crucial mediator of muscle atrophy in catabolic states. Localized overexpression of IGF-I in tibialis anterior (TA) muscle was performed by injection of IGF-I cDNA followed by electroporation 3 d before starting dexamethasone injections (0.1 mg/kg.d sc). A control plasmid was electroporated in the contralateral TA muscle. Dexamethasone induced atrophy of the TA muscle as illustrated by reduction in muscle mass (403 +/- 11 vs. 461 +/- 19 mg, P < 0.05) and fiber cross-sectional area (1759 +/- 131 vs. 2517 +/- 93 mum(2), P < 0.05). This muscle atrophy was paralleled by a decrease in the IGF-I muscle content (7.2 +/- 0.9 vs. 15.7 +/- 1.4 ng/g of muscle, P < 0.001). As the result of IGF-I gene transfer, the IGF-I muscle content increased 2-fold (15.8 +/- 1.2 vs. 7.2 +/- 0.9 ng/g of muscle, P < 0.001). In addition, the muscle mass (437 +/- 8 vs. 403 +/- 11 mg, P < 0.01) and the fiber cross-sectional area (2269 +/- 129 vs. 1759 +/- 131 mum(2), P < 0.05) were increased in the TA muscle electroporated with IGF-I DNA, compared with the contralateral muscle electroporated with a control plasmid. Our results show therefore that IGF-I gene transfer by electroporation prevents muscle atrophy in glucocorticoid-treated rats. Our observation supports the important role of decreased muscle IGF-I in the muscle atrophy caused by glucocorticoids.

The physical basis of nucleic acid base stacking in water.

It has been argued that the stacking of adenyl groups in water must be driven primarily by electrostatic interactions, based upon NMR data showing stacking for two adenyl groups joined by a 3-atom linker but not for two naphthyl groups joined by the same linker. In contrast, theoretical work has suggested that adenine stacking is driven primarily by nonelectrostatic forces, and that electrostatic interactions actually produce a net repulsion between adenines stacking in water. The present study provides evidence that the experimental data for the 3-atom-linked bis-adenyl and bis-naphthyl compounds are consistent with the theory indicating that nonelectrostatic interactions drive adenine stacking. First, a theoretical conformational analysis is found to reproduce the observed ranking of the stacking tendencies of the compounds studied experimentally. A geometric analysis identifies two possible reasons, other than stronger electrostatic interactions, why the 3-atom-linked bis-adenyl compounds should stack more than the bis-naphthyl compounds. First, stacked naphthyl groups tend to lie further apart than stacked adenyl groups, based upon both quantum calculations and crystal structures. This may prevent the bis-naphthyl compound from stacking as extensively as the bis-adenyl compound. Second, geometric analysis shows that more stacked conformations are sterically accessible to the bis-adenyl compound than to the bis-naphthyl compound because the linker is attached to the sides of the adenyl groups, but to the ends of the naphthyl groups. Finally, ab initio quantum mechanics calculations and energy decompositions for relevant conformations of adenine and naphthalene dimers support the view that stacking in these compounds is driven primarily by nonelectrostatic interactions. The present analysis illustrates the importance of considering all aspects of a molecular system when interpreting experimental data, and the value of computer models as an adjunct to chemical intuition.

A proposed architecture for ambulatory systems development.

We have developed an architecture and application framework known as the Ambulatory Services Architecture (ASA) for computerized management of patient records for ambulatory care. Our primary design goals included the development of an architecture that will be readily adaptable to advances in technology needed to enhance computerized patient record systems (e.g., multimedia, human-computer interfaces such as voice-to-text, and a fully distributed objects, etc.) and a data model and database implementation capable of providing the flexibility and extensibility needed to support a broad spectrum of specialty medical practices. This report describes the data model, access control, and the client/server components of the Ambulatory Services Architecture.

Collaborative social and medical service application.

Baylor College of Medicine has five Teen Health Clinics (THC) dispersed throughout Harris county. The population served by the clinics includes inner-city adolescent boys and girls 19 years of age and under. Patients receive services such as family planning, sexually transmitted disease screening and treatment, perinatal care, counseling, and support services. Adolescents may receive services at any one of the clinics at no cost to the adolescent or their dependents. Given the geographical distribution of the clinics and the reliance on paper-based records, client services cannot be provided efficiently or expeditiously. According to the statistics developed by Clinic staff, ineffective coordination of service needs and client schedules undermine the follow-up needed for effective care. For example, a counselor will often need to balance a school schedule, clinic visits, well baby follow-up, and the Best Friends Program for a new mother. In addition, the lack of ready access to patient information impairs the ability of clinical and social service staff to provide continuity of care. In fact, some cases of client dropout are attributable to these difficulties. We have developed the Collaborative Social and Medical Service Application (CSMSA) to facilitate the provision of social and medical services to this population. The CSMSA is a domain-specific application based on a robust infrastructure known as the Ambulatory Services Architecture (ASA). This system is designed to support integrated social and ambulatory care. The ASA is a Baylor developed application framework and architecture for the computerization of the patient medical record in the ambulatory care setting. The working environment for the CSMSA user is an integrated desktop which provides an operating environment for both third-party applications and the CSMSA, as well as a fundamental set of services. The integrated desktop services include a mechanism for object organization or grouping, a facility for the management of desktop objects including disposal and storage, and an embedded search utility to assist in the location of desktop objects as well as other application objects. The access control mechanism will provide the security for the desktop environment by requiring the user to log into and out of the environment. This security mechanism will also enable/disable CSMSA tools based on the user's role at the clinic. The CSMSA is designed to provide work process, functionality and data access appropriate to the responsibilities of the user, e.g., a THC clerk may have permission to view a patient's orders but does not have the facility to create an order. Patient context is defined through a patient browser containing the master patient index or a user defined patient list. Patient encounters are managed using forms based on the process for each encounter type (e.g., initial visit). Forms are used for data entry and for queries. Data entry forms are only committed to the patient database (i.e., making the patient data available to other users who have permission to access the data) when the user approves the data. The CSMSA provides a means to suspend an entry into a chart by saving incomplete or non-committed data entry forms as part of the desktop state which is restored when the user logs into the system. CSMSA was designed to provide a robust, expandable application capable of accommodating the changes in health care and social services delivery while encompassing evolving software standards and new technology. This goal was achieved using object-oriented methodologies and technologies, combined with an object-oriented database management system as the foundation of our server to facilitate the evolution of our data model.

The development of a client application for the collaborative social and medical services system.

This paper describes the design and implementation of a client application for the Baylor College of Medicine Teen Health Clinics. The application is the front end to the Collaborative Social and Medical Services System (CSMSS) under development by Baylor's Medical Informatics and Computing Research Program [8]. The application provides distributed access to an underlying object oriented database system. A process driven and patient centered design will provide staff members with a complete set of services, including forms for data entry and viewing, query, and access management to facilitate efficient and effective delivery of services. Role-specific interfaces will be supplied for clerks, nurses, nurse practitioners, physicians, and social workers. The client application is being designed using object oriented methodologies and technologies with the C++ programming language, and will operate within a Microsoft Windows operating environment utilizing Object Linking and Embedding for application interoperability.

Collaborative Social and Medical Service System.

This paper describes the Collaborative Social and Medical Services System, a robust information infrastructure for integrated social and medical care. The Collaborative Social and Medical Services System design and architecture address the primary goals of creating a readily extensible social and ambulatory care system. Our initial step toward reaching this goal is the delivery of an application supporting the operations of the Baylor Teen Health Clinics. This paper discusses our prototype experiences, system architecture, components, and the standards we are addressing.

The MEDLINE Retriever.

Baylor College of Medicine has developed the MEDLINE Retriever, a tool to query MEDLINE, the data-base of medical literature at the National Library of Medicine. The MEDLINE Retriever communicates via the Internet to achieve excellent response time for MEDLINE queries. It uses the X Window System and the Motif toolkit, and employs the Knowbot Operating Environment developed by the Corporation for National Research Initiatives. We discuss the architecture of the MEDLINE Retriever, focusing on the graphical user interface that we have developed, as well as our experiences in developing and deploying the MEDLINE Retriever at Baylor. The MEDLINE Retriever is an extension of Baylor's IAIMS design concept that brought forth the Virtual Notebook System, and fits well with Baylor's aims with regard to the High Performance Computing Initiative.

Octopus photoreceptor membranes. Surface charge density and pK of the Schiff base of the pigments.

The chromophore of octopus rhodopsin is 11-cis retinal, linked via a protonated Schiff base to the protein backbone. Its stable photoproduct, metarhodopsin, has all-trans retinal as its chromphore. The Schiff base of acid metarhodopsin (lambda max = 510 nm) is protonated, whereas that of alkaline metarhodopsin (lambda max = 376 nm) is unprotonated. Metarhodopsin in photoreceptor membranes was titrated and the apparent pK of the Schiff base was measured at different ionic strengths. From these salt-dependent pKs the surface charge density of the octopus photoreceptor membranes and the intrinsic Schiff base pK of metarhodopsin were obtained. The surface charge density is sigma = -1.6 +/- 0.1 electronic charges per 1,000 A2. Comparison of the measured surface charge density with values from octopus rhodopsin model structures suggests that the measured value is for the extracellular surface and so the Schiff base in metarhodopsin is freely accessible to protons from the extracellular side of the membrane. The intrinsic Schiff base pK of metarhodopsin is 8.44 +/- 0.12, whereas that of rhodopsin is found to be 10.65 +/- 0.10 in 4.0 M KCl. These pK values are significantly higher than the pK value around 7.0 for a retinal Schiff base in a polar solvent; we suggest that a plausible mechanism to increase the pK of the retinal pigments is the preorganization of their chromophore-binding sites. The preorganized site stabilizes the protonated Schiff base with respect to the unprotonated one. The difference in the pK for the octopus rhodopsin compared with metarhodopsin is attributed to the relative freedom of the latter's chromophore-binding site to rearrange itself after deprotonation of the Schiff base.

Regeneration of bovine and octopus opsins in situ with natural and artificial retinals.

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.