A mycobacterial ABC transporter mediates the uptake of hydrophilic compounds.

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis1-3. Although Mtb can synthesize vitamin B12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis2. Mtb does not encode any characterized cobalamin transporter4-6; however, the gene rv1819c was found to be essential for uptake of cobalamin1. This result is difficult to reconcile with the original annotation of Rv1819c as a protein implicated in the transport of antimicrobial peptides such as bleomycin7. In addition, uptake of cobalamin seems inconsistent with the amino acid sequence, which suggests that Rv1819c has a bacterial ATP-binding cassette (ABC)-exporter fold1. Here, we present structures of Rv1819c, which reveal that the protein indeed contains the ABC-exporter fold, as well as a large water-filled cavity of about 7,700 Å3, which enables the protein to transport the unrelated hydrophilic compounds bleomycin and cobalamin. On the basis of these structures, we propose that Rv1819c is a multi-solute transporter for hydrophilic molecules, analogous to the multidrug exporters of the ABC transporter family, which pump out structurally diverse hydrophobic compounds from cells8-11.

Non-dispensing pharmacists integrated into general practices as a new interprofessional model: a qualitative evaluation of general practitioners' experiences and views.

BACKGROUND: A new interprofessional model incorporating non-dispensing pharmacists in general practice teams can improve the quality of pharmaceutical care. However, results of the model are dependent on the context. Understanding when, why and how the model works may increase chances of successful broader implementation in other general practices. Earlier theories suggested that the results of the model are achieved by bringing pharmacotherapeutic knowledge into general practices. This mechanism may not be enough for successful implementation of the model. We wanted to understand better how establishing new interprofessional models in existing healthcare organisations takes place. METHODS: An interview study, with a realist informed evaluation was conducted. This qualitative study was part of the Pharmacotherapy Optimisation through Integration of a Non-dispensing pharmacist in primary care Teams (POINT) project. We invited the general practitioners of the 9 general practices who (had) worked closely with a non-dispensing pharmacist for an interview. Interview data were analysed through discussions about the coding with the research team where themes were developed over time. RESULTS: We interviewed 2 general practitioners in each general practice (18 interviews in total). In a context where general practitioners acknowledge the need for improvement and are willing to work with a non-dispensing pharmacist as a new team member, the following mechanisms are triggered. Non-dispensing pharmacists add new knowledge to current general practice. Through everyday talk (discursive actions) both general practitioners and non-dispensing pharmacists evolve in what they consider appropriate, legitimate and imaginable in their work situations. They align their professional identities. CONCLUSIONS: Not only the addition of new knowledge of non-dispensing pharmacist to the general practice team is crucial for the success of this interprofessional healthcare model, but also alignment of the general practitioners' and non-dispensing pharmacists' professional identities. This is essentially different from traditional pharmaceutical care models, in which pharmacists and GPs work in separate organisations. To induce the process of identity alignment, general practitioners need to acknowledge the need to improve the quality of pharmaceutical care interprofessionally. By acknowledging the aspect of interprofessionality, both general practitioners and non-dispensing pharmacists will explore and reflect on what they consider appropriate, legitimate and imaginable in carrying out their professional roles. TRIAL REGISTRATION: The POINT project was pre-registered in The Netherlands National Trial Register, with Trial registration number NTR-4389.

Effects of non-dispensing pharmacists integrated in general practice on medication-related hospitalisations.

AIMS: To evaluate the effect of non-dispensing pharmacists (NDPs) integrated in general practice on medication-related hospitalisations, drug burden index and costs in patients at high risk of medication problems (being 65 years or older and using 5 or more chronic medications). METHODS: This was a multicentre, nonrandomised, controlled intervention study with pre-post comparison (2013 vs June 2014 to May 2015) in 25 general practices in the Netherlands, comparing NDP-led care (intervention) with 2 current pharmaceutical care models (usual care and usual care plus). In the intervention group, 10 specially trained NDPs were employed in general practices to take integral responsibility for the pharmaceutical care. They provided a broad range of medication therapy management services both on patient level (e.g. clinical medication review) and practice level (e.g. quality improvement projects). In the control groups, pharmaceutical care was provided as usual by general practitioners and community pharmacists, or as usual plus, when pharmacists were additionally trained in performing medication reviews. RESULTS: Overall, 822 medication-related hospitalisations were identified among 11 281 high-risk patients during the intervention period. After adjustment for clustering and potential confounders, the rate ratio of medication-related hospitalisations in the intervention group compared to usual care was 0.68 (95% confidence interval: 0.57-0.82) and 1.05 (95% confidence interval: 0.73-1.52) compared to usual care plus. No differences in drug burden index or costs were found. CONCLUSIONS: In general practices with an integrated NDP, the rate of medication-related hospitalisations is lower compared to usual care. No differences with usual care plus were found.

Successful treatment for subinvolution of placental sites in the bitch with low oral doses of progestagen.

Subinvolution of placental sites (SIPS) is the major cause of persistent sanguineous vaginal discharge after parturition in the bitch. Spontaneous remission is common but may take several months, and hence, medical therapy to end the discharge is often requested. In this retrospective study, we evaluated the effect of treatment for SIPS with low oral doses of a progestagen. Nine bitches with SIPS, but otherwise clinically healthy, were found in the computer database of the Department of Clinical Sciences of Companion Animals. Seven of these bitches were treated with low oral doses of a progestagen (megestrol acetate, 0.1 mg/kg body weight (bw) once daily for the 1st week, then 0.05 mg/kg bw once daily for the 2nd week). The other two bitches were untreated. Treatment results were evaluated by a telephone questionnaire. Progestagen treatment was successful in all of the treated dogs; sanguineous vaginal discharge stopped within the treatment period. One of the two untreated dogs remained symptomatic until the next oestrus, approximately 120 days after parturition, and the other remained symptomatic until 6 weeks before the start of the next pro-oestrus, 270 days after parturition. No side effects of the progestagen treatment were observed. Subsequent gestations, parturitions and puerperal periods of 5 mated bitches were uneventful. One bitch did not become pregnant after mating. In conclusion, the results of this study indicate that oral administration of low doses of progestagen for 2 weeks is effective in stopping persistent sanguineous vaginal discharge in bitches with SIPS, with neither side effects nor reduced subsequent fertility.

Progesterone receptor isoforms in the mammary gland of cats and dogs.

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

Minimal external masculinization in a SRY-negative XX male Podenco dog.

Normal mammalian sex differentiation takes place in three genetically controlled steps: chromosomal sex determination (XX or XY), gonadal differentiation and development of the phenotypic sex. Animals are considered to be sex reversed if chromosomal sex determination and gonadal development are not in agreement. In this report, sex reversal is described in a 1.5-year-old Podenco dog that was referred because of suspected recurrent growth of a previously removed os clitoridis in the vulva. With that exception the dog was phenotypically female, but had never been in oestrus and exhibited male behaviour. Abdominal ultrasonography showed a small tubular structure dorsal to the bladder, consistent with a uterus. An ovoid structure resembling a gonad was visible between the right kidney and inguinal canal. Plasma testosterone concentrations before and after GnRH administration indicated the presence of functional testicular tissue. Two testes, each with its epididymis and ductus deferens, and a complete bicornuate uterus were removed surgically. Cytogenetic analysis of peripheral blood lymphocytes showed a normal female karyotype (78, XX). These findings are consistent with the diagnosis of an XX male. PCR analysis of genomic DNA revealed that the SRY gene was absent. In summary, this report describes the first SRY-negative XX male Podenco dog with an almost complete female phenotype despite high basal and stimulated plasma testosterone concentrations. It is hypothesized that the clinical observations in this dog may have been caused by reduced and delayed Müllerian-inhibiting substance secretion and the absence of conversion of testosterone to dihydrotestosterone due to 5alpha-reductase deficiency.

Hormonal changes in spontaneous and aglépristone-induced parturition in dogs.

To increase our understanding of the endocrine changes associated with parturition in dogs, plasma concentrations of progesterone (P4), 15-ketodihydroprostaglandin F(2alpha) (PGFM), estradiol-17-beta (E2beta), cortisol, ACTH, prolactin (PRL), LH, and FSH were measured in six spontaneously whelping bitches and in six bitches in which parturition was induced with the progesterone-receptor blocker aglépristone on day 58 of pregnancy. Expulsion of pups in the induced group took place in the presence of P4 concentrations that were still elevated. PGFM concentrations increased before parturition in both groups, but levels were lower in the induced bitches. PGFM levels reached a maximum in both groups during parturition and quickly decreased in the spontaneously whelping group after parturition, but remained elevated in the induced group. In both groups, cortisol concentrations reached similar maximum levels during the last 30 h before the onset of expulsion. During the 3 days postpartum, cortisol concentrations were higher in the induced group. The highly variable ACTH concentrations did not differ significantly throughout the study within or between groups. In both groups, E2beta concentrations decreased and PRL concentrations increased between the late gestational period and the 30-h period before parturition. Concentrations of both LH (spontaneously whelping group) and FSH (both groups) decreased between late gestation and the postpartum period. The results of this study illustrate the hormonal changes around parturition in the bitch, and reveal that aglépristone-induced parturition is associated with still incomplete luteolysis, an altered PGFM profile, and elevated postpartum cortisol concentrations as compared with spontaneously whelping dogs.

Physiology of the canine anoestrus and methods for manipulation of its length.

Progression from early to late anoestrus is characterized by the appearance of a larger number of gonadotrophin-releasing hormone (GnRH) pulses with a higher amplitude, an increase in the sensitivity of the pituitary to GnRH, an increase in ovarian responsiveness to gonadotrophins, and an increase in basal plasma follicle-stimulating hormone (FSH) concentration. A period of increased luteinizing hormone (LH) pulsatility has been observed shortly before the onset of pro-oestrus. Apart from these changes in the hypothalamus-pituitary-ovary axis, the initiation of a new follicular phase in the bitch is also stimulated by dopaminergic influences other than the accompanying plasma prolactin decrease. Metergoline, a drug which in a low dosage lowers the plasma prolactin concentration via a serotonin-antagonistic pathway, does not shorten the anoestrus; while bromocriptine, in a dosage insufficient to cause a decrease in the plasma prolactin concentration, does prematurely induce a follicular phase. These observations indicate that it is not the decrease in the plasma prolactin concentration, but another dopamine-agonistic influence that plays a crucial role in the transition to a new follicular phase. The dopamine-agonist induced oestrus is associated with a rapid rise in the basal plasma FSH concentration, similar to what is observed during the physiological late anoestrus. Administration of GnRH, eCG and oestrogens may also be used to induce oestrus but with variable results. Oestrus can be prevented surgically or medically, for which purpose progestagens are the most important drugs. The mechanism is still unclear, although it has been demonstrated that with continuing medroxyprogesterone acetate (MPA) treatment the FSH response to GnRH stimulation decreases and changes occur in the pulsatile release of the gonadotrophins. In general, LH pulses coincide with a FSH pulse, but during MPA treatment, LH pulses were observed while there was such a small increase in FSH that it was not recognized as significant FSH pulse.

Estriolum treatment in the bitch: a risk for uterine infection?

Purulent vaginal discharge in a bitch in which ovariohysterectomy has been performed is often caused by inflammation of the uterine stump. The inflammation is due to either cystic endometrial hyperplasia (CEH) induced primarily by progesterone from remnant ovarian tissue or exogenous progestagens, or it is due to the presence of unabsorbed suture material. This report describes a 9-year-old Irish setter with hemopurulent vaginal discharge and non-pruritic symmetrical alopecia, which had undergone ovariohysterectomy 3.5 years ago and which had been treated with estriolum daily for the past 2.5 years because of urinary incontinence. Vaginoscopy revealed hemopurulent discharge throughout the vagina and vestibule. Cytological examination of ultrasound-guided fine-needle aspiration biopsies of a large mass in the hypogastricum, which appeared to be the uterine cervical stump, revealed septic purulent inflammation. The concentration of plasma progesterone was low and the concentration of plasma 17-ss oestradiol did not increase after gonadotrophin-releasing hormone administration. No remnant ovarian tissue was found by abdominal ultrasonography, laparotomy, or histological examination of mesovarian pedicles. Laparotomy revealed uterine stump empyema. Histological examination of the surgically removed mass excluded both CEH and unabsorbed suture material as the cause of the stump empyema. Instead, it is hypothesized that the long-term treatment with estriolum was a causative factor. This suggests that bitches treated with estriolum should be examined regularly.

Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM.

Uptake of vitamin B12 is essential for many prokaryotes, but in most cases the membrane proteins involved are yet to be identified. We present the biochemical characterization and high-resolution crystal structure of BtuM, a predicted bacterial vitamin B12 uptake system. BtuM binds vitamin B12 in its base-off conformation, with a cysteine residue as axial ligand of the corrin cobalt ion. Spectroscopic analysis indicates that the unusual thiolate coordination allows for decyanation of vitamin B12. Chemical modification of the substrate is a property other characterized vitamin B12-transport proteins do not exhibit.

In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

Comparison of urine and oral fluid as matrices for screening of thirty-three benzodiazepines and benzodiazepine-like substances using immunoassay and LC-MS(-MS).

Benzodiazepines are the most frequently detected medicinal drugs in drivers. The use of benzodiazepines is associated with an increased road accident risk. In this study, the presence of benzodiazepines detected by liquid chromatography-(tandem) mass spectrometry [LC-MS(-MS)] in oral fluid and urine samples obtained from drivers stopped during a roadside survey was compared. In addition, the sensitivity and selectivity of enzyme multiplied immunoassay technique (EMIT II Plus) relative to LC-MS(-MS) was determined for both matrices. A total number of 1,011 urine samples were collected and screened for benzodiazepines using immunoassay (IA) (EMIT II Plus; cutoff 300 ng/mL). In the IA-positive (n = 25) and a group of randomly selected negative urine samples (n = 79), the presence or absence of benzodiazepines was confirmed by LC-MS-MS after deglucuronidation. The corresponding oral fluid samples (n = 101, 3 samples omitted), were analyzed by LC-MS(-MS) and IA (EMIT II Plus; cutoff 10 ng/mL). The presence of benzodiazepines was demonstrated by LC-MS-(MS) in all IA-positive urine samples, but in only four corresponding oral fluid samples. Concentrations in oral fluid were, one substance excepted, lower than in urine. The sensitivity and specificity of EMIT II Plus were better by using urine as matrix for screening of benzodiazepines than by using oral fluid. The results show that benzodiazepines are detectable in oral fluid. More research has to be done to determine the pharmacokinetic profile of the different benzodiazepines in oral fluid and to study the relationship between dose, concentration (in oral fluid and blood), and impairment.

Temporal relations between plasma concentrations of luteinizing hormone, follicle-stimulating hormone, estradiol-17beta, progesterone, prolactin, and alpha-melanocyte-stimulating hormone during the follicular, ovulatory, and early luteal phase in the bitch.

Compared with other domestic animals, relatively little is known about the changes in, and temporal relations between, reproductive hormones around the time of ovulation in the domestic bitch. Therefore, plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17beta, progesterone, prolactin (PRL), and alpha-melanocyte-stimulating hormone (alpha-MSH) were determined one to six times daily from the start of the follicular phase until 5 days after the estimated day of ovulation in six Beagle bitches. In all bitches, the pre-ovulatory LH surge was accompanied by a pre-ovulatory FSH surge. A pre-ovulatory PRL or alpha-MSH surge was not observed. The pre-ovulatory FSH and LH surges started concomitantly in four bitches, but in two bitches the FSH surge started 12 h earlier than the LH surge. The FSH surge (110+/-8 h) lasted significantly longer than the LH surge (36+/-5 h). In contrast with the pre-ovulatory FSH surge, the pre-ovulatory LH surge was bifurcated in four of six bitches. The mean plasma LH concentrations before (1.9+/-0.4 microg/L) and after (1.9+/-0.3 microg/L) the LH surge were similar, but the mean plasma FSH concentration before the FSH surge (1.6+/-0.3 U/L) was significantly lower than that after the FSH surge (3.1+/-0.2 U/L). In most bitches the highest plasma estradiol-17beta concentration coincided with or followed the start of the pre-ovulatory LH surge. In five of the six bitches the plasma progesterone concentration started to rise just before or concurrently with the start of the LH surge. In conclusion, the results of this study provide evidence for the differential regulation of the secretion of LH and FSH in the bitch. In addition, the interrelationship of the plasma profiles of estradiol-17beta and LH suggests a positive feedback effect of estradiol-17beta on LH surge release. The start of the pre-ovulatory LH surge is associated with an increase in the plasma progesterone concentration in this species.

Differential regulation of the secretion of luteinizing hormone and follicle-stimulating hormone around the time of ovulation in the bitch.

Plasma concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were determined 3-6 times daily in six Beagle bitches from the start of the follicular phase until 5 d after the estimated day of ovulation. The aim of the study was to gain more detailed information regarding the changes in and the temporal relation between these hormones around the time of ovulation. In all bitches, the pre-ovulatory LH surge was accompanied by a pre-ovulatory FSH surge. The mean duration of the pre-ovulatory FSH surge (110 +/- 8 h) was significantly longer than that of the pre-ovulatory LH surge (36 +/- 5 h). The FSH surge started concomitantly with the pre-ovulatory LH surge in four bitches, and 12 h before the start of the LH surge in the other two bitches. The pre-ovulatory LH surge had a bifurcated pattern in four bitches. The mean plasma LH concentration before (1.9 +/- 0.4 microg/L) and after (1.9 +/- 0.3 microg/L) the pre-ovulatory LH surge were similar. The mean plasma FSH concentration during the period 72-28 h before the pre-ovulatory LH surge (1.6 +/- 0.3 U/L) was lower (P < 0.001) than that during the period 100-144 h after the pre-ovulatory LH surge (3.1 +/- 0.2U/L). In conclusion, this study demonstrated concurrent pre-ovulatory surges of FSH and LH and provided more evidence for differential regulation of the secretion of FSH and LH.

Validation of a noninvasive diagnostic tool to verify neuter status in dogs: The urinary FSH to creatinine ratio.

Determining the presence of functional gonadal tissue in dogs can be challenging, especially in bitches during anestrus or not known to have been ovariectomized, or in male dogs with nonscrotal testes. Furthermore, in male dogs treated with deslorelin, a slow-release GnRH agonist implant for reversible chemical castration, the verification of complete downregulation of the hypothalamic-pituitary-gonadal (HPG) axis can be difficult, especially if pretreatment parameters such as the size of the testes or prostate gland are not available. The aims of this study were to validate an immunoradiometric assay for measurement of FSH in canine urine, to determine if the urinary FSH to creatinine ratio can be used to verify the neuter status in bitches and male dogs, as an alternative to the plasma FSH concentration, and to determine if downregulation of the HPG axis is achieved in male dogs during deslorelin treatment. Recovery of added canine FSH and serial dilutions of urine reported that the immunoradiometric assay measures urinary FSH concentration accurately and with high precision. Plasma FSH concentrations (the mean of two samples, taken 40 minutes apart) and the urinary FSH to creatinine ratio were determined before gonadectomy and 140 days (median, range 121-225 days) and 206 days (median, range 158-294 days) after gonadectomy of 13 bitches and five male dogs, respectively, and in 13 male dogs before and 132 days (median, range 117-174 days) after administration of a deslorelin implant. In both bitches and male dogs, the plasma FSH concentration and the urinary FSH to creatinine ratio were significantly higher after gonadectomy, with no overlapping of their ranges. Receiver operating characteristic analysis of the urinary FSH to creatinine ratio revealed a cut-off value of 2.9 in bitches and 6.5 in males to verify the presence or absence of functional gonadal tissue. In male dogs treated with deslorelin, the plasma FSH concentrations and urinary FSH to creatinine ratios were significantly lower after administration of the implant, but their ranges overlapped. We conclude that the urinary FSH to creatinine ratio can be used to verify the neuter status of bitches and male dogs. However, it cannot be used for the assessment of complete downregulation of the HPG axis after administration of a deslorelin implant. The urinary FSH to creatinine ratio is preferable over the plasma FSH concentration because it involves only one sample that can be collected relatively easy and noninvasively.

The effects of kisspeptin agonist canine KP-10 and kisspeptin antagonist p271 on plasma LH concentrations during different stages of the estrous cycle and anestrus in the bitch.

Kisspeptin (KP) plays a key role in the regulation of the hypothalamic-pituitary-gonadal axis via the release of GnRH. As normal KP signaling is essential for reproductive function, it could be an interesting new target for therapeutic interventions, e.g., nonsurgical contraception in dogs. The aims of the present study were to investigate the effect of KP-10 administration on plasma LH concentration in different stages of the reproductive cycle and to investigate the suitability of p271 as KP antagonist in the bitch. Two groups of six adult Beagle bitches were used. In one group, plasma LH concentration was determined before (40 and 0 minutes) and 10, 20, 40, and 60 minutes after the intravenous administration of 0.5-µg/kg body weight (BW) canine KP-10. In the other group, the bitches received a continuous intravenous infusion with p271 (50 µg/kg BW/h) for 3 hours, and 0.5-µg/kg BW canine KP-10 was administered intravenously 2 hours after the start of the p271 infusion. Their plasma LH concentration was determined before (-40 and 0 minutes) and 30, 60, 90, 120, 130, 140, 160, and 180 minutes after the start of the p271 infusion. In both groups, the experiments were performed during the follicular phase, the first and second half of the luteal phase, and during anestrus. Canine KP-10 induced an increase of plasma LH concentration during all estrous cycle stages and anestrus. There was no difference in LH response between the two groups. The lowest LH response was seen during the follicular phase and the highest response during anestrus. The area under the curve (AUC) for LH and LH increment in the follicular phase were lower than those in anestrus. The AUC LH and LH increment in the first half of the luteal phase were lower than those in the second half of the luteal phase and anestrus. The AUC LH and LH increment in the second half of the luteal phase were not different from those in anestrus. Continuous administration of the antagonist p271 did not alter basal plasma LH concentration and could not prevent or lower the LH response to KP-10 in any of the cycle stages and anestrus. It can be concluded that the LH response to KP-10 is dependent on estrous cycle stage and that peripheral administrated p271 cannot be used as KP antagonist in the dog. This provides new insight in reproductive endocrinology of the bitch, which is important when KP signaling is considered for therapeutic interventions, such as for estrus induction or nonsurgical contraception in the bitch.

Phosphatidylinositol as the endogenous activator of the (Na+ + K+)-ATPase in microsomes of rabbit kidney.

Incubation of rabbit kidney microsomes with pig pancreatic phospholipase A2 produced residual membrane preparations with very low (Na+ + K+)-ATPase activity. The activity could be restored by recombination with lipid vesicles of negatively-charged glycerophospholipids. Vesicles of pure phosphatidylcholine and phosphatidylethanolamine were virtually inactive in this respect, but could reactivate in the presence of cholate. Incubation of the microsomes with a combination of phospholipase C (Bacillus cereus) and spingomyelinase C (Staphylococcus aureus) resulted in 90--95% release of the phospholipids. The residual membrane contained only phosphatidylinositol and still showed 50--100% of the (Na+ + K+)-ATPase activity.

The interaction of spectrin-actin and synthetic phospholipids. II. The interaction with phosphatidylserine.

Sonicated vesicles of phosphatidylserine and phosphatidylserine/phosphatidylcholine mixtures were recombined with spectrin-actin from human erythrocyte ghosts. Morphological properties and physicochemical characteristics of the recombinates were studied with freeze etch electron microscopy, 31P NMR and differential scanning calorimetry. Sonicated dimyristoyl phosphatidylserine vesicles show a decrease in enthalpy change of the lipid phase transition upon addition of spectrin-actin. These vesicles collapse and fuse, into multilamellar structures in the presence of spectrin-actin, as demonstrated by freeze fracturing and NMR. Spectrin-actin cannot prevent the salt formation between phosphatidylserine and Ca2+, all phosphatidylserine is withdrawn from the lipid phase transition. In contrast a protection against the action of Mg2+ could be observed. Mixed bilayers of dimyristoyl phosphatidylserine/dimyristoyl phosphatidylcholine show phase separations at molar ratios above 1/1 (van Dijck, P.W.M., de Kruijff, B., Verkleij, A.J., van Deenen, L.L.M. and de Gier, J. (1978) Biochim. Biophys. Acta 512, 84--96). These phase spearations can be prevented by spectrin-actin. Ca2+-induced lateral phase separations in cocrystallizing phosphatidylserine/phosphatidylcholine mixtures, can be reduced by spectrin-actin. Formation of the Ca2+-phosphatidylserine salt, occurring in addition to lateral phase separation when mixtures contain more than 30 mol % phosphatidylserine, cannot be prevented by spectrin-actin.

Differential miscibility properties of various phosphatidylcholine/lysophosphatidylcholine mixtures.

Using enthalphy data from differential scanning calorimetry experiments and 13C-NMR linewidths of specifically (N-Me-13C)-labelled lipids, the miscibility properties of phosphatidylcholines and lysophosphatidylcholines in liposomal dispersons have been investigated. It was found that 16 : 0 lysophosphatidylcholine mixes homogeneously in 16 : 0/16 : 0 phosphatidylcholine bilayers. Mixtures of 16 : 0 lysophosphatidylcholine with 18 : 1c/18 : 1c phosphatidylcholine, of 18 : 1c lysophosphatidylcholine with 16 : 0/16 : 0 phosphatidylcholine and of 18 : 1c lysophosphatidylcholine with 18 : 1c/18 : 1c phosphatidylcholine of exhibited immiscibility in the phosphatidylcholine gel state.

The role of albumin conformation in the binding of diazepam to human serum albumin.

The effect of hydrogen, chloride and calcium ions on the binding of diazepam to human serum albumin has been studied by circular dichroism and equilibrium dialysis. In all cases the molar ellipticity of the diazepam-albumin complex increases with pH over the pH range 5 to 9. Under these conditions the free concentration of diazepam at a constant low drug to protein ratio decreases with pH. This free concentration is higher in the presence of chloride and calcium ions. With a two state conformational model for albumin it was shown, that the pH dependences of molar ellipticity of the diazepam-albumin complex and of the free concentration of diazepam are linked. It was demonstrated that the N-B transition of albumin is involved in the pH dependent binding of diazepam. The consequences of these findings for equilibrium dialysis procedures in determining free plasma levels of diazepam are discussed.