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Progesterone (Pro) is a potent neurosteroid and promotes recovery from moderate Traumatic Brain Injury but its clinical application is severely impeded by its poor water solubility. Here we demonstrate that reversibly binding Pro within hydrophobically modified hyperbranched polyglycerol (HPG-Cn-MPEG) enhances its solubility, stability and bioavailability. Synthesis, characterization and Pro loading into HPG-Cn-MPEG is described. The release kinetics are correlated with structural properties and the results of Differential Scanning Calorimetry studies of a family of HPG-Cn-MPEGs of varying molecular weight and alkylation. While the maximum amount of Pro bound correlates well with the amount of alkyl carbon per molecule contributing to its hydrophobicity, the dominant first order rate constant for Pro release correlates strongly with the amount of structured or bound water in the dendritic domain of the polymer. The results provide evidence to justify more detailed studies of interactions with biological systems, both single cells and in animal models.

Zusammenfassung Die Epithelzellen des Rattennebenhodens wurden durch Verdauung des kleingeschnittenen Nebenhodens mit Pronase und anschliesend mit Zusatz von Kollagenase plus Hyaluronidase freigesetzt. Die Epithelzellen wurden sodann von Zellverunreinigungen durch differenzierte Zentrifugierung und Sedimentierung getrennt. Die isolierten Zellen blieben lebensfahig, wie durch den Ausschlus von Trypanblau bestatigt werden konnte. Die Zellen atmeten in Gegenwart von Glukose und die Respirationsrate war bei der folgenden Zugabe von Pyruvate nicht verandert. Resumen Las celulas epiteliales del epididimo se liberaron de epididimo troceado por digestion con pronasa, seguida de colagenasa mas hialuronidasa. Posteriormente, se separaron las celulas epiteliales de las celulas contaminantes mediante centrifugacion diferencial y sedimentacion a unidad de gravedad a traves de un gradiente de sucrosa en Ringer. Las celulas aisladas permanecian viables como se comprobo por la exclusion del azul tripon. Las celulas respiraban en presencia de glucosa y no se altero la respiracion por la adicion subsiguiente de piruvato.

We wanted to investigate the visual outcome of horses presented with iris prolapse and treated with corneal transplantation.To evaluate the visual outcome of horses with iris prolapse treated with penetrating keratoplasty alone and penetrating keratoplasty in combination with overlying conjunctival or amniotic membrane grafting.A retrospective medical records study of horses presented to the University of Florida Veterinary Medical Center for iris prolapse and treated with penetrating keratoplasty in the period of 1998-2010. Data collected from the medical records included signalment, clinical descriptions of ocular lesions, treatments, and therapeutic outcome.Iris prolapses in this study were caused by corneal ulcers with keratomalacia (n = 37). All horses were treated medically for infection, hyperproteinase activity and iridocyclitis, and then surgically treated with either penetrating keratoplasty alone (n = 9) or penetrating keratoplasty with either a conjunctival pedicle flap (n = 22), amniotic membrane transplant (n = 5) or amnion membrane and conjunctival pedicle flap (n = 1). The eyes were visual postoperatively in a majority of the cases (n = 24; 64.9%). Limited vision was noted in 6 eyes (16.2%), 3 eyes became phthisical (8.1%) and 4 globes were enucleated (10.8%). Graft rejection manifested as some degree of donor corneal graft opacification in all cases. Anterior synechiae were present in 48.6% of the eyes. Wound dehiscence and aqueous humour leakage were also common as post operative problems.Penetrating keratoplasty alone or in combination with an overlying graft of conjunctiva or amniotic membrane can achieve a successful visual outcome in a high percentage of horses with iris prolapse.

The widespread availability of medications and herbal products on the Internet has increased the potential for poisonings. We are reporting a case of mild, acute lithium toxicity occurring after the intentional misuse of a lithium-containing "dietary supplement" (Find Serenity Now) obtained over the Internet.An 18-year-old woman presented to our emergency department (ED) after ingesting 18 tablets of Find Serenity Now; each tablet contained, according to the listing, 120 mg of lithium orotate [3.83 mg of elemental lithium per 100 mg of (organic) lithium orotate compared to 18.8 mg of elemental lithium per 100 mg of (inorganic) lithium carbonate]. The patient complained of nausea and reported one episode of emesis. Her examination revealed normal vital signs. The only finding was a mild tremor without rigidity. Almost 90 minutes after the ingestion, her serum lithium level was 0.31 mEq/L, a urine drug screen was negative, and an electrocardiogram (ECG) showed a normal sinus rhythm. The patient received intravenous fluids and an anti-emetic; one hour later, her repeat serum lithium level was 0.40 mEq/L. After 3 hours of observation, nausea and tremor were resolved, and she was subsequently transferred to a psychiatric hospital for further care. Prior human and animal data have shown similar pharmacokinetics and shared clinical effects of these lithium salts.Over-the-Internet dietary supplements may contain ingredients capable of causing toxicity in overdose. Chronic lithium toxicity from ingestion of this product is also of theoretical concern.

To describe and evaluate the use of corneoconjunctival transposition as a surgical treatment for corneal sequestra in cats.Fifteen cats (17 eyes) of various breeds and ages with corneal sequestra treated surgically with corneoconjunctival transposition.Retrospective medical records study.Median age was 39 months. Eight patients were castrated males, five were spayed females, and two were intact females. The left eye was affected in eight cats, the right eye was affected in five cats, and both eyes were affected in two cats. Mean (+/- SD) sequestrum diameter was 4.4 +/- 2.4 mm (range 1-10 mm) and the sequestrum was located centrally in all cases. Sequestra occurred in the superficial stroma in 8/17 corneas, in the anterior one third of the stroma in 3/17 corneas, and in the middle one third of the stroma in 6/17 corneas. Median duration of clinical signs prior to presentation was 39 days (range 14-1095 days). Median time to healing, defined as the number of days from surgery to cessation of medical treatment, was 34 days (range 25-86 days). There were no recurrences and minimal scar formation.Corneoconjunctival transposition is a valid procedure for surgical treatment of corneal sequestra in cats. The procedure results in excellent cosmesis and functional vision with no recurrences.

The objective of this study was to evaluate the possible relationship between the administration of parenteral and/or oral [corrected] enrofloxacin and the onset of acute retinal degeneration in cats. The animals studied included 17 cats that received systemic enrofloxacin and developed retinal degeneration soon thereafter.In this retrospective clinical study, cats that received parenteral and/or oral [corrected] enrofloxacin and developed acute blindness were identified. Parameters recorded included breed, age, sex, enrofloxacin dosage (daily dose and number of days administered), medical condition for which the antibiotic had been prescribed, ophthalmic signs, examination results, and the visual outcome. Fundus photographs were obtained in seven cats, and electroretinography was performed in five cats. Histopathology was performed on two eyes from one cat (case 1) that received enrofloxacin 5 months previously and developed retinal degeneration.All cats were the domestic shorthair breed; seven were females (one neutered) and ten were males (seven castrated). Ages ranged from 3 to 16 years old (mean +/- SD; 8.8 +/- 4.6 years). The medical disorders for which enrofloxacin was administered ranged from lymphoma and pancreatitis to otitis and dermatitis, and eight cats had urinary diseases. The daily and total dosage of enrofloxacin and number of days of administration were also highly variable. Presenting clinical signs were most often mydriasis and acute blindness. All cats had diffuse retinal degeneration as evidenced by increased tapetal reflectivity and retinal vascular attenuation. Absence of recordable electroretinographic responses suggested diffuse and extensive outer retinal disease. Vision returned in a few cats, but the retinal degeneration persisted or even progressed. Histopathology of two eyes revealed primarily outer retinal degeneration, with diffuse loss of the outer nuclear and photoreceptor layers, and hypertrophy and proliferation of the retinal pigment epithelium.Parenteral and/or oral [corrected] enrofloxacin is potentially retinotoxic in some cats, and may result in acute and diffuse retinal degeneration. Blindness often results, but some cats may regain vision. Practitioners should adhere closely to the manufacturer's current enrofloxacin dosage recommendation (5 mg/kg q 24 h), and continue clinical observations for this drug toxicity in cats.

The viscosity of whole blood measured at low shear rates is determined partly by shear resistance of the red cell aggregates present, stronger aggregation increasing the viscosity in the absence of other changes. Effects of cell deformability can confound interpretation and comparison in terms of aggregation, however, particularly when the plasma viscosity is high. We illustrate the problem with a comparison of hematocrit-adjusted blood from type 1 diabetes patients and controls in which it is found the apparent and relative viscosities at a true shear rate of 0.20 s-1 are lower in the patient samples than age matched controls, in spite of reports that aggregation is increased in such populations. Because the plasma viscosities of the patients were higher on average than controls, we performed a series of experiments to examine the effect of plasma protein concentration and viscosity on normal blood viscosity. Dilution or concentration by ultrafiltration of autologous plasma and viscosity measurements at low shear on constant hematocrit red cell suspensions showed (a) suspension viscosity at 0.25 and 3 s-1 increased monotonically with plasma protein concentration and viscosity but (b) the relative viscosity increased, in concert with the microscopic aggregation grade, up to a viscosity of approximately 1.25 mPa-s but above this the value the relative viscosity no longer increased as the degree of aggregation increased in concentrated plasmas. It is suggested that in order to reduce cell deformation effects in hyperviscous pathological plasmas, patient and control plasmas should be systematically diluted before hematocrit is adjusted and rheological measurements are made. True shear rates should be calculated. Comparison of relative viscosities at low true shear rates appears to allow the effects of red cell aggregation to be distinguished by variable shear rate viscometry in clinical blood samples.

The surface-induced mineralization (SIM) technique was used to produce hydroxyapatite (HAP) coatings on external fixation pins with the antimicrobial agent, chlorhexidine, incorporated within the coating. The SIM process involved surface modification of the substrate with organic functional groups followed by immersion in aqueous supersaturated calcium phosphate solutions. X-ray diffraction spectra confirmed that hydroxyapatite coatings were formed. Chlorhexidine was incorporated into the coating by placing the substrate into various chlorhexidine solutions in between mineralization cycles. Total uptake was measured by dissolution of the coating into a 0.1 M nitric acid solution and measuring the chlorhexidine concentration using UV spectroscopy at 251 nm. Release rates were measured by submersion of coated substrates into saline solutions and measuring chlorhexidine UV absorbency at 231 nm as a function of time. Results show an initial rapid release followed by a period of slower sustained release. The anti-microbial efficacy of the HAP-chlorhexidine coatings was evaluated in vitro using a Staphylococcus aureus cell culture. Initial results show a large "inhibition zone" formed around the chlorhexidine/HAP coating vs. coatings with HAP only. This preliminary work clearly demonstrates that SIM HAP coatings have great potential to locally deliver antimicrobial agents such as chlorhexidine at implantation sites, which may greatly reduce the incidence of pin tract infection that occurs in external fixation.

Studies on such systems as the lens of the eye and theoretical considerations suggest that phase separation may well occur in cytoplasm. In this chapter, several issues relevant to this question are raised. It is suggested that while the interaction between water and the macromolecules in a mixture is proving crucial to their phase separation behavior, the abnormal water that is widely observed in cytoplasm and concentrated protein solutions is unlikely to constitute a thermodynamic phase in the sense of phase separation studies. The role of fixed structures in the cytoplasm, the likelihood that the volume of separated phases would be small and subject to spreading over the fixed structures and the expectation that much of the phase volume could be occupied and dominated by properties of the interface are also discussed. Finally, some experimental approaches to studying the existence of liquid-liquid phases in cytoplasm are proposed. While there is no proof that phase separation exists in cytoplasm, application of some of the techniques outlined might well provide more positive evidence for its presence.

Incompatible pairs of polymers separate into two phases in aqueous solution above a few percentage points total concentration. Protein pairs can also produce phase separation, but at somewhat higher concentrations. In this chapter, we explore the effect of high background concentrations of macromolecules on phase separation of pairs of species which would not be at sufficiently high concentration to separate in the absence of the uninvolved species. Effects produced by such high background concentrations are known as macromolecular crowding. Dramatic enhancements in various association reactions due to crowding have been predicted and observed but its effects on phase separation in biological mixtures typical of the cytoplasm have not been examined. Here, we describe a calculation based on the Flory-Huggins treatment of concentrated polymer solutions that sheds some light on this issue. We find that a background of 20 wt % of a high molecular weight species greatly reduces the concentrations needed to produce phase separation in a mixture of two incompatible macromolecules if one is more hydrophobic than the other. Given the high total concentration of macromolecules in cytoplasm, it is perhaps surprising that phases have not been observed. This issue is discussed and some explanations offered.

To investigate the distribution of intraocular pressure (IOP) and cup properties in a colony of rhesus monkeys that has had no outside genetic input since 1938 (approximately 12 generations). This sample of sequestered monkeys is significantly larger than any previously reported. Comparisons are made with a sample of random-source monkeys to develop population estimates defining the limits of normalcy.The IOP and cup/disc ratio estimates were collected from 701 eyes of 354 adult rhesus monkeys from the closed colony on Cayo Santiago, Puerto Rico. Results for IOP were compared with the normal rhesus IOP population distribution function calculated from an earlier sample of genetically heterogeneous rhesus.The mean +/- standard deviation IOP in the Cayo Santiago monkeys (15.8+/-3 mmHg) related well to the calculated "normal" rhesus distribution (14.5+/-2 mmHg) below and around the mean IOP only. Above the mean rhesus IOP, the samples from the Cayo monkeys were strongly skewed: 129 eyes had IOP more than two standard deviations above the normal mean IOP, and 54 eyes had IOP more than three standard deviations above the normal mean IOP. Cup/disc ratio estimations tended to cluster as higher values in the higher IOP quartiles. Some eyes with IOP below the mean had cup/disc ratios0.5. Values for IOP that were more than two standard deviations above the mean and cup/disc ratios0.4 were not uniformly distributed across social groupings, although incidence of high IOP was more than 25% in one group.After 12 generations with the same genetic pool, expression of ocular hypertension and large optic disc cups is high but not uniform.

A new antibody to lysergic acid diethylamide (LSD) was used to develop a novel indirect ELISA for the quantification of drug in urine. Evaluation of the new assay with the commercially available LSD ELISA (STC Diagnostics) shows improved performance. The test requires 50 microL of urine, which is used to measure concentrations of drug in the microg/L to ng/L range. The limit of detection was 8 ng/L compared with 85 ng/L in the commercial assay, and analytical recoveries were 98-106%. Our test detected 0.1 microg/L of LSD in urine with an intraassay CV of 2.4% (n = 8) compared with 6.0% for a 0.5 microg/L sample in the commercial assay (n = 20). The upper and lower limits of quantification were estimated to be 7 microg/L and 50 ng/L, respectively. Specificity was evaluated by measuring the extent of cross-reactivity with 24 related substances. Drug determination using the new assay offers both improved sensitivity and precision compared with existing methods, thus facilitating the preliminary quantitative estimation of LSD in urine at lower concentrations with a greater degree of certainty.

The authors identify any diurnal intraocular pressure (IOP) variation in healthy Cayo Santiago macaques, as well as Cayo Santiago macaques with normotensive and hypertensive primary open-angle glaucoma (POAG), to further evaluate their potential value as a model for human POAG.Twenty-four monkeys (eight animals each of the healthy control, normotensive glaucoma, and hypertensive glaucoma groups) were sedated with ketamine hydrochloride and the IOP measured hourly from 8:00 AM until 3:00 PM with a Tonopen XL applanation tonometer (Mentor, Norwell, MA, U.S.A.). Mean IOP time profiles, mean IOPs at each observation time, and linear trends in mean IOP over time were compared among groups. Intraocular pressure rate-of-change over time and residual variation about the regression line for each individual eye were analyzed. Cup/disc ratio (C/D) was compared with baseline IOP, IOP profile mean and slope, and IOP residual variation.A significant group and time effect on mean IOP (p = 0.0001 and 0.011, respectively), with highest values at 2:00 PM or 3:00 PM, and a significant increasing linear trend in mean IOP over time in the hypertensive group were observed (p = 0.012). Intermediate readings between control and hypertensive mean IOPs were identified for the normotensive glaucoma group. The biggest range of IOP variation was found in the hypertensive glaucoma monkeys. Higher variations in IOP did not lead to an increase in C/D.This study provides evidence that the Cayo Santiago macaques are a valuable model for human normotensive and hypertensive POAG.

To evaluate in vitro susceptibility to topical antifungal medications, as measured by minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC50%), of fungal isolates from horses with ulcerative keratomycosis in Florida; to compare results with those of other studies to identify differences in susceptibility patterns among fungi isolated from horses in different geographic regions; and to note indications of fungal resistance to drugs tested in other studies.Corneal fungal cultures from client-owned horses from Florida with ulcerative keratomycosis (n = 22).Fungal cultures were plated on Emmons modified Sabouraud dextrose agar and mycobiotic agar, examined weekly for growth, and kept for a total of 30 days. In vitro MIC and IC50% of fluconazole, itraconazole, ketoconazole, miconazole, and natamycin were measured for each fungal isolate.Aspergillus (n = 9; 41%), Fusarium (7; 32%), Penicillium (2; 9%), Cylindrocarpon (1; 4%), Scytalidium (1; 4%), and Torulopsis (1; 4%) spp and an unidentified yeast (1; 4%) were isolated. Fungi were most susceptible to antifungal drugs in the following order: natamycin and miconazole equally, itraconazole, and ketoconazole, although no significant difference was found among drugs. Fungi were significantly less susceptible to fluconazole (P0.0001) than to the other 4 drugs.Initial antifungal therapy with topically applied natamycin, miconazole, itraconazole, or ketoconazole is recommended for ulcerative keratomycosis in horses in the subtropical environment of Florida.Specific antifungal treatment of horses with ulcerative keratomycosis should be based on history, results of ophthalmic examination, cytologic findings, isolation of the pathogenic fungus, and known prevalence of unique ocular fungi in specific geographic areas. In vitro antifungal susceptibility testing may be most beneficial in aiding documentation of pharmacologic susceptibility patterns of fungi in specific geographic regions.

To determine whether photoreceptor outer segments can be found in aqueous humor from dogs with rhegmatogenous retinal detachment (RRD).Case series.4 dogs with unilateral RRD, 2 dogs with bilateral RRD, 1 dog with unilateral non-RRD, and 1 dog with glaucoma without retinal detachment.Aqueous humor samples were fixed in 2% glutaraldehyde and examined by means of transmission electron microscopy.Outer segments were found in aqueous humor from 7 of 8 eyes with RRD but were not found in aqueous humor from dogs with non-RRD or glaucoma.Photoreceptor outer segments may move into the anterior chamber of eyes with RRD.

To analyze the vitreal amino acid concentrations in dogs with breed-related primary glaucoma to determine whether excitotoxic amino acids associated with retinal genglion cell death in other species were present in affected dogs.11 normal control and 10 glaucomatous canine eyes.Amino acid analyses were performed by high-pressure liquid chromatography in masked manner.Eyes from dogs with primary glaucoma had significantly high vitreal glutamate concentration, compared with values for eyes of clinically normal control dogs. Mean (+/-SD) glutamate concentrations were 31.7 +/- 12.4 and 6.9 +/- 6.3 microM in glaucomatous and normal eyes, respectively (P0.0001). Eyes from dogs with glaucoma also had lower vitreal glycine (37.0 +/- 17.0 vs 59.4 +/- 28.2 microM; P0.043) and higher of vitreal tryptophan (39.0 +/- 22.8 vs 17.5 +/- 11.2 microM; P0.012) concentrations, compared with values for normal eyes.Glutamate concentration potentially toxic to retinal ganglion cells is associated with the pathogenesis of primary glaucoma in dogs. Increased glutamate concentration provides evidence of an ischemic mechanism for retinal ganglion cell death and optic nerve atrophy in dogs with glaucoma.The emphasis on reduction and normalization of high intraocular pressure as the primary focus of treatment for glaucoma in dogs should be augmented by other therapeutic approaches.

Size-dependent exclusion of macromolecules from gel matrices has long been discussed in terms of pore models. An alternate approach is to calculate the partition coefficient of the distributed species between the matrix material and the mobile phase assuming that the gel can be treated as a polymer solution of appropriate concentration and molecular weight. This approach is particularly appealing in attempting to predict the behaviour of 'tentacle' phases in which the matrix contains anchored linear neutral polymers. The mean field theory of polymer solution is used to predict K, the partition coefficient of a polymer molecule distributing between the gel and mobile phases. The reduction in entropy suffered by the macromolecule in the gel phase is sufficient to produce an exponential dependence of K on the molecular weight of the partitioning species. The enthalpy of interaction between the gel polymer and the distributed species provides a parameter which describes the specificity or recognition in the interaction. The predicted linear dependence of in K on protein molecular weight is satisfactorily borne out by a data set on eight standard proteins chromatographed on four separate gel types.

To document the incidence of postoperative ocular hypertension (POH) after cataract surgery in dogs.Retrospective analysis of medical records.88 dogs that had had cataract surgery.The effect of several categorical variables on the development of POH was evaluated statistically. Postoperative ocular hypertension was defined as intraocular pressure25 and30 mm of Hg.The incidence of POH25 mm of Hg was 48.9%;30 mm of Hg, 33.8%;40 mm of Hg, 20.1%; and50 mm of Hg, 5.8%. Mean onset of POH25 mm of Hg was 4.9 hours. The incidence of POH was not affected by the type of surgery. Eyes that had phacoemulsification developed POH significantly more rapidly (mean, 3.9 hours), compared with those that had extracapsular lens extraction (8.4 hours). Mean phacoemulsification duration was greater in eyes that developed POH, and older dogs were more likely to develop POH. Development of POH was not correlated with sex, stage of cataract, type of surgical procedure performed, intraocular lens placement, preoperative lens-induced uveitis, or posterior lens capsule tears and vitrectomy. However, eyes that received intraocular lens implants developed POH more rapidly, compared with eyes without implants.The high incidence and early onset of POH after cataract surgery suggests that routine use of antiglaucoma medications in the first 12 hours after surgery is warranted.

Using an applanation tonometer, 5 replicate intraocular pressure (IOP) measurements were obtained from each eye of 12 young clinically normal, American alligators. Alligator length ranged from 46 to 117 cm, measured from snout to tail tip. All IOP were recorded by a single observer at an ambient temperature of approximately 25 C, and ranged from 5 to 35 mm of Hg. Observer reliability was excellent (intraclass r = 0.93), and IOP did not change over the ordered sequence of 5 replicate measurements/eye. Replicate IOP) measurements were, therefore, averaged in each eye for comparison between eyes of the same alligator. Left and right eve IOP were highly correlated within individual alligators (r = 0.92), whereas the mean within animal difference between left and right eye IOP was not statistically significant (95% confidence interval [CI] for the left eye-right eye mean difference, - 1.9 to 1.3 min of Hg). Mean IOP determined for 5 confirmed females and 3 confirmed males did not differ significantly between the sexes (95% CI for the male-female difference in means, -2.1 to 3.7 mm of Hg). Mean +/- SEM IOP of 23.7 + 2.1 mm of Hg determined for 4 alligators-50 cm long was significantly (P = 0.009) greater than mean IOP of 11.6 + 0.5 mm of Hg determined for 8 alligators50 cm long (95% CI for the difference in means, 8.5 to 15.7 mm of Hg). In young alligators, the relation between body length and IOP appears to be nonlinear, possibly with a negative exponent.

Mean (+/- SD) value for results of the Schirmer tear test without use of topical anesthesia on 142 rabbit eyes was 5.30 (+/- 2.96) mm/min; range was 15 mm/min, with low value of 0 and high value of 15 mm/min. Two SD from the mean provided a normal clinical range of 0 to 11.2 mm/min for inclusion of 95% of the population. Significant differences were not evident between eye (right vs left) or gender or among breeds; interaction among eye, gender, and breed also was not observed. Although mean values for 2 breeds were significantly different from values in the other 10 breeds and from each other, they were still within the proposed normal range of 2 SD. The Schirmer tear test without topical anesthesia may be useful in rabbits for evaluation of increased values correlated with ocular irritation, rather than for determination of decreased values associated with keratoconjunctivitis sicca.

Oscillatory potentials (OP) and electroretinograms (ERG) were recorded from clinically normal dogs after 5, 10, 15, 20, 30, 40, 50, and 60 minutes of dark adaptation. At the end of the adaptation period, OP were characterized by 5 distinct positive peaks, O1 through O5, with mean latencies of 14.46, 20.24, 27.38, 35.31, and 44.85 ms, respectively, and with mean amplitudes ranging from 7.20 to 34.84 microV. After 60 minutes of dark adaptation, the ERG had a mean a-wave latency of 12.03 ms and a mean b-wave amplitude of 109.29 microV. Peaks O3 and O4, which partially mask the summit of the b-wave, had mean latencies of 28.66 and 36.83 ms, respectively. The mean amplitude of the b-wave measured to the peak of O3 was 240.06 microV and 230.73 microV when measured to peak O4. Changes in the OP during dark adaptation consisted of significant (P less than 0.05) increases in the latencies of O1, O2, and O3, and significant increases in the amplitudes of O1, O3, O4, and O5. Concurrent ERG changes consisted of significant increases in the amplitudes of the a-wave and b-wave measured from O3 and O4, and significant increases in the latencies of peaks O3 and O4 on the b-wave.

Five horses with severe nonulcerative keratouveitis had corneal lesions characterized by a pink stromal infiltrate that initially appeared in the stroma near the limbus. Unremitting iridocyclitis also was evident. In 3 horses, microscopic lesions consisted of marked corneal stromal fibrosis, with mild to severe inflammatory cellular infiltration. Corticosteroids and mydriatic/cycloplegics applied topically and corticosteroids and nonsteroidal anti-inflammatory medications administered parenterally were used with varying degrees of success to control pain and retain vision.

Four unrelated cockatiels had various degrees of maldevelopment of the eyelids. Only 1 eye of the 8 was normal. The condition had features compatible with ankyloblepharon or cryptophthalmos. Attempts were made to surgically establish an artificial palpebral fissure. Despite extensive surgical intervention and postoperative topical administration of corticosteroids, the skin over the orbit reverted to the preoperative state in all birds. Anecdotal information along with our findings suggest that this condition, although uncommon, is widespread in the cockatiel population in the United States.

Cell partitioning in two-phase aqueous polymer systems was used to examine hydrophobic and surface charge-related membrane properties of fresh and cryopreserved human blood phagocytes. This technique is highly sensitive to cell surface characteristics, and the partition behavior depends exponentially on the membrane properties involved. The transition from fresh to cryopreserved and reconstituted cells was accompanied by a significant loss of net negative charge without detectable alteration in hydrophobic membrane properties as detected by the partition technique. The partition coefficient (PC), which is the proportion of cells partitioning into the upper phase, when measured for fresh cells mixed with dimethyl sulfoxide (Me2SO) and cryopreserved leukocytes. No difference was detected between the PCs of the total leukocytes and phagocytes as determined by differential leukocyte counts of the upper polymer phase. The significantly reduced PC of cells prepared by dextran (Dx) separation in both charge-sensitive and -insensitive systems is attributable to the capacity of Dx to adsorb, in part irreversibly, to cells so that those carrying Dx tend to partition with Dx in the lower phase. These results serve to illustrate the utility of partitioning as a highly sensitive method to probe leukocyte surface membrane properties.

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (35S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein-specific in that only selected proteins became associated with the cells. Binding was not cell-specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.

SUMMARY The influence of steroids on the specific activities of glycolytic enzymes was studied in the caput and cauda epididymidis of the rat. In castrated animals, increases in enzyme activities were induced by the administration of androgenic steroids whilst oestradiol and progesterone were without effect. On the other hand, the 'antiandrogenic' steroid, cyproterone acetate (16 mg/kg per day for 2 weeks) did not cause a decrease in enzyme activities when administered to animals with ligated efferent ducts. Not all glycolytic enzymes responded to androgens, but more enzymes responded in the caput than in the cauda epididymidis. Administration of testosterone propionate to castrated animals demonstrated that maximum enzyme activity was produced at a dose of 0·1 mg/kg per day whilst higher doses were required to achieve maximum weight of the epididymis, seminal vesicles and prostate. Following castration of animals with ligated efferent ducts, no changes in enzyme activities were observed for approximately 2 days, but then activities declined over the next 2 weeks. When testosterone propionate was administered to castrated animals, a similar lag of approximately 2 days occurred before enzyme activities began to increase.

Summary: Application of a viscometric assay to the haemagglutination induced by Aeromonas salmonicida strain 438 showed that shear forces can enhance the strength of bacterial adhesion. The D-mannose/L-fucose-sensitive reaction proceeded in two phases, an initial phase in which the degree of aggregation remained constant during shearing and a second stage, induced by shear, in which agglutination was enhanced as shear was maintained. The results strongly paralleled those found in studies of concanavalin A-induced haemagglutination, providing good evidence that adhesion in this species took place via lectin-like molecules. Methyl-α-D-mannoside, which strongly inhibits haemagglutination in this system, would not fully reverse the shear-dependent reaction. EGTA inhibited and reversed both phases, however. The effects of bacterial concentration, temperature, time of growth, pH, and a spectrum of monosaccharide inhibitors were also studied. The results demonstrated that the shear-dependent reaction has a number of features which distinguish it from the initial stage of haemagglutination, implying differences in the underlying biochemical mechanisms involved.

From the Lifshitz theory, Hamaker coefficients have been computed for all the monoglyceride-alkane thin films in polar liquids described in the previous two parts of this paper. The calculations have been based on three different models of the films. The first model represents the film simply as an isotropic layer of liquid hydrocarbon, in which it is assumed that the lipid and solvent chains are intimately mixed. The second is similar to the first except that it includes two 0.4 nm layers of glycerol to represent the glyceride polar groups of the lipid. The theoretical Hamaker coefficients for these two models are identical to withinca. 10% and, for twelve out of the twenty-four systems examined, they agree to withinca. 20 % with the corresponding experimental coefficients. In the remaining systems, which mainly involve very thin films, the theoretical and experimental results differ by as much as a factor of three. In seeking an explanation for these discrepancies, calculations were carried out for the third model in which, instead of assuming complete mixing of the lipid and solvent chains, it is assumed that they are completely segregated. The hydrocarbon layer is thus split into three layers, i. e. lipid chains–alkane solvent–lipid chains. As with the first and second models, for about half the systems, theory and experiment agree to within 20 % but, for the remaining films, the third model is a considerable improvement as it yields no discrepancies with the experimental data greater than a factor of two. Such discrepancies as do still exist may be accounted for by recognizing thatsomesolvent must in­evitably be present in the lipid chain regions and that, in fact, the state of the interior of a film is intermediate to those assumed in the second and third models. The results exemplify well how successful the Lifshitz theory can be in liquid systems, but they also emphasize the very large effects which small differences in dielectric properties can have on the van der Waals forces in thin films.

Free energies of formation of “black” lipid films have been determined from their contact angles. In these films the repulsive forces are of such short range that it is possible to estimate relatively accurately that part of the free-energy change that originates from the van der Waals forces. It is shown that if the van der Waals free energies are interpreted on the assumption that the films are isotropic layers of hydrocarbon bounded by semi-infinite aqueous phases, the Hamaker coefficients vary consideraly from one film to another, contrary to the predictions of the Lifshitz theory. If, on the other hand, it is recognized that the hydrocarbon region of a film is, in fact, a layered structure and that there are differences, albeit small, between the dielectric properties of the chains of the lipid stabilizer and the alkane solvent (some of which is retained in the film), the conflict between theory and experiment is largely removed. Thus, Hamaker coefficients calculated from the Lifshitz theory for multilayered systems agree well in nearly all instances with the corresponding experimental coefficients.

Carnitine and glycerylphosphorylcholine, two well-known constituents of the mammalian epididymis, are shown to be equally characteristic of another sperm-storing organ, namely the spermatophore of a cephalopod mollusc, Octopus dofleini martini .

Adult sheep which has been castrated either before or after puberty were treated with a variety of steroids. The administration of testosterone propionate, oestrone, oestradiol-17 beta or diethylstilboestrol to animals castrated before puberty caused them to mount oestrous ewes. Oestradiol-17 alpha was less effective than these hormones in this regard, whilst oestriol, hexoestrol and 5 alpha-dihydrotestosterone were ineffective. The response to oestradiol-17 beta was not altered by the concurrent administration of dexamethasone to block the pituitary-adrenal axis which suggests that oestradiol-17 beta was not exerting its effect indirectly by causing the release of adrenal steroids. When 5 alpha-dihydrotestosterone was administered in conjunction with oestradiol-17 beta intromission and ejaculation were observed in addition to mounting behaviour. When rams were castrated as adults their mating behaviour slowly declined over the course of 2 years. After this time, mounting behaviour was rapidly restored by the administration of oestradiol-17 beta but not by 5 alpha-dihydrotestosterone. These results are consistent with the hypothesis that oestrogens are the ultimate agents responsible for promoting mating behaviour in male animals and hence aromatizable androgens, such as testosterone, are effective whereas non-aromatizable androgens, such as 5 alpha-dihydrotestosterone, are not.

The incorporation of radioactive mannose and fucose into secretory glycoproteins by rat epididymal tissue was studied using tissue pieces in vitro. The appearance of radioactive macromolecular products in the medium occurred after a lag phase of 2 h with radioactive mannose, but with radioactive fucose the lag phase was only 15 min. Preincubation of tissue for 2 h before the addition of radioactive mannose increased the subsequent rate of incorporation by reducing the lag phase from 2 to 1 h. Tunicamycin reduced the incorporation of radioactive mannose and fucose into macromolecular products to approximately 15 and 50% of normal in the caput and cauda respectively; maximum inhibition required 10 micrograms tunicamycin/ml in the caput and 2 micrograms/ml in the cauda. Reduction of radioactive sugar incorporation by tunicamycin did not result in qualitative changes in the profile of the radioactive glycoproteins that were secreted. However, immunoprecipitation of proteins D and E from incubations with radioactive methionine or mannose revealed that tunicamycin caused these proteins to be synthesized and secreted in a non-glycosylated form. Prior castration of animals reduced the incorporation of radioactive mannose and fucose, and qualitative changes in the profiles of secreted radioactive glycoproteins were apparent.

The proteins of epididymal luminal fluid and of spermatozoa recovered from different regions of the rat epididymis were examined by polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions. Albumin (A) and four major pre-albumin bands (B-E) were observed in epididymal fluid from the cauda on non-denaturing gels. By comparing the migration of these bands with that of standard globular proteins on denaturing gels, the molecular weight of Bands B and C was estimated to be 16 000, Band D was 30 000 and Band E was 32 000. Bands D and E were apparently glycoproteins since they stained with periodic acid-Schiff's reagent and were bound by an affinity column of Concanavalin A. The pre-albumin proteins (B-E) were of epididymal origin since they (a) were not detected in blood serum, (b) were not detected in testicular extracts and (c) were still found after ligation of the efferent ducts. From the incorporation of radioactive methionine, Bands B and C were shown to be synthesized in the initial segment and caput. The regional distribution of luminal proteins indicated that protein D was added in the caput and cauda and protein E in the cauda. This regional origin of luminal proteins was confirmed by the altered protein profiles consequent upon the reduced fluid flow through the epididymis brought about by ligation of the efferent ducts. The androgen-dependence of epididymal protein synthesis was also investigated using radioactive methionine. Castration had little effect on total protein synthesis but resulted in the specific reduction of the synthesis of proteins B and C. Several changes were observed in the relative amounts of specific proteins extracted from spermatozoa from different regions of the epididymis and several of these proteins had molecular weights identical with those in luminal fluid. However, there was no evidence for any substantial binding to spermatozoa of the pre-albumin proteins (B-E) of luminal fluid.

Eight units of blood were drawn into modified CPD containing 25 per cent higher glucose and 17.3 mg adenine (0.25 mM in blood). Red blood cell concentrates (RCC) were prepared to a mean hematocrit (Hct) of 70, the cells stored at 4 C, and plasma adenine and red blood cell adenosine triphosphate (ATP) were measured weekly for 42 days. The removal of plasma in the preparation of RCC reduced by 39 per cent the available adenine. As a result measurable plasma adenine was depleted by 21 days. The loss of ATP in RCC occurs at a significantly faster rate than in whole blood stored under the same conditions. When red blood cells are stored at higher HCT or for periods longer than 35 days, increased anticoagulant adenine levels are recommended.

1. Procedures were developed for the extraction and assay of glycolytic enzymes from the epididymis and epididymal spermatozoa of the rat. 2. The epididymis was separated into four segments for analysis. When rendered free of spermatozoa by efferent duct ligation, regional differences in enzyme activity were apparent. Phosphofructokinase, glycerol phosphate dehydrogenase and glucose 6-phosphate dehydrogenase were more active in the proximal regions of the epididymis, whereas hexokinase, lactate dehydrogenase and phosphorylase were more active in the distal segment. These enzymes were less active in the epididymis of castrated animals and less difference was apparent between the proximal and distal segments. However, the corpus epididymidis from castrated rats had lower activities of almost all enzymes compared with other epididymal segments. 3. Spermatozoa required sonication to obtain satisfactory enzyme release. Glycolytic enzymes were more active in spermatozoa than in epididymal tissue, being more than 10 times as active in the case of hexokinase, phosphoglycerate kinase and phosphoglycerate mutase. 4. The specific activities of a number of enzymes in the epididymis were dependent on the androgen status of the animal. These included hexokinase, phosphofructokinase, aldolase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, glycerol phosphate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphorylase. 5. The caput and cauda epididymidis differed in the extent to which enzyme activities changed in response to an altered androgen status. The most notable examples were hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, 6-phosphogluconate dehydrogenase and phosphorylase.

1. Enzyme activities (units/g wet wt.) were determined in the caput and cauda epididymidis and in epididymal spermatozoa of the rat. 2. The activity of most enzymes in the cauda was between 50 and 100% of that in the caput, except that ATP citrate lyase was barely detectable in the cauda. 3. Spermatozoa, unlike epididymal tissue, contained sorbitol dehydrogenase but lacked ATP citrate lyase. NADP+-malate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, succinate dehydrogenase, carnitine acetyltransferase and citrate synthase were 5 to 400 times as active in spermatozoa as in epididymal tissue. 4. 2-Oxoglutarate dehydrogenase was the least active member of the tricarboxylic acid cycle in all tissues and most closely matched the measured flux through the cycle. 5. The concentrations of hydroxyacyl-CoA dehydrogenase and carnitine palmitoyltransferase were equivalent to the more active enzymes of the tricarboxylic acid cycle, indicating the capacity for extensive lipid oxidation, and the presence of 3-hydroxybutyrate dehydrogenase suggests that these tissues can also oxidize ketone bodies. 6. Transfer of reducing equivalents from cytoplasm to mitochondrion is unlikely to occur by means of the glycerol phosphate cycle because mitochondrial glycerol 3-phosphate dehydrogenase is relatively inactive in epididymal tissue, whereas the cytoplasmic enzyme has little activity in spermatozoa, but transfer may be accomplished by the malate-aspartate shuttle. 7. Transfer of acetyl units from mitochondrion to cytoplasm could be effected by the pyruvate-malate cycle in the caput of androgen-maintained rats, but not in the other tissues because of the low activity of ATP citrate lyase. Acetyl unit transfer could take place via acetylcarnitine, mediated by carnitine acetyltransferase. 8. Castration resulted in a decrease in the concentration of nearly all enzymes, although subsequent administration of testosterone restored concentrations to values similar to those in animals maintained by endogenous androgen. The extent to which enzyme concentration was changed by an alteration in androgen status was highly variable, but was most marked in the case of pyruvate carboxylase.

The concentration of total carnitine (i.e. carnitine plus acetylcarnitine) was measured in seminal plasma and spermatozoa of men and rams. In ram semen, there was a close correlation between the concentration of spermatozoa and that of total carnitine in the seminal plasma, indicating that the epididymal secretion was the sole source of seminal carnitine. The percentage of total carnitine present as acetylcarnitine was 40% in seminal plasma and 70-80% in spermatozoa. The acetylation state of carnitine in seminal plasma was apparently not influenced by the metabolic activity of spermatozoa in ejaculated ram semen as no change was found in the plasma concentration of carnitine or acetylcarnitine up to 45 min after ejaculation. In spermatozoa, the activity of carnitine acetyltransferase (EC 2.3.1.7) was approximately equivalent to that of carnitine palmitoyltransferase (EC 2.3.1.21); and the activity of these enzymes was similar in ram and human spermatozoa but greater in rat spermatozoa. It is concluded that there is no correlation between the content of either total carnitine or the carnitine acyltransferases and the respiratory capacity of spermatozoa.

Two acidic secretory epididymal glycoproteins, protein D of 27,000 daltons and protein E of 28,000 daltons, have been purified and antisera prepared against each separately. Both proteins were found to share common immunological determinants when tested by double immunodiffusion and tandem crossed immunoelectrophoresis. By selective immunoprecipitation, protein D was shown to be synthesized and secreted by all regions of the epididymis with the exception of the initial segments. In contrast, the synthesis and secretion of protein E was restricted to the corpus and proximal cauda.

The availability of glycerol in the epididymal lumen, and the extent of its utilization by spermatozoa, was studied by measurement of the substrate concentration in epididymal fluid and its oxidation in vitro. The source of luminal glycerol was sought by examining its penetration through the epididymal epithelium of a sperm-free tubule by the technique of luminal perfusion during intravenous infusion of glycerol. [3H]Glycerol was rapidly metabolized to a volatile and mobile molecule that quickly equilibrated between blood plasma and the epididymal lumen and so could not be used to monitor transfer. When blood levels of glycerol were raised by infusion of large amounts of the unlabelled compound, glycerol was detected in epididymal perfusates and a positive linear correlation existed between the rate of secretion into the epididymal lumen and the blood plasma concentration, suggesting that passive diffusion across the epithelium from blood could occur. In normal rats, however, the concentration of glycerol in sperm-free epididymal fluid (1.15 mM) exceeded that in blood plasma (0.35 mM). Luminal glycerol is therefore thought to arise from degradation of epididymal lipid.

1. A method is described for the extraction, purification and separation of acid-soluble phosphorus compounds from mammalian semen. [8-14C]ATP and [8-14C]AMP were used as internal recovery standards to measure the breakdown and loss of these nucleotides in the procedure. 2. Bull, ram, boar and stallion semen was separated into seminal plasma and spermatozoa and the two fractions were examined separately. The overall composition of the mixture of the phosphorus compounds extracted from the two fractions was similar for the four species. 3. Glycerylphosphorylcholine and glycerylphosphorylinositol were the two phosphorus compounds identified in extracts of seminal plasma. ATP, ADP, AMP, GTP, GDP, NAD, fructose 1,6-diphosphate and glucose 6-phosphate were identified in extracts prepared from spermatozoa.