Effect of activators and inhibitors on human blood platelets: study by freeze-fracturing technique and electron microscopy

Samples of normal human platelet-rich plasma were activated by ADP (0.5 microM and 20 microM) or by cold treatment (4 degrees C). The effects of incubation in EDTA, colchicine or cytochalazin B were also studied. Samples were prepared by the freeze-fracturing technique and examined by electron microscopy. Faces of fractured membranes were examined for aggregation of particles, for pore-openings in the membrane, and for the general morphological appearance of the membrane-fractured faces. No aggregation of particles was found in platelet-rich plasma preparations treated with ADP, EDTA, colchicine or cytochalazin B, nor did the pore openings reveal any changes following these treatments. We conclude that platelet activation and an increase in microviscosity of the platelet membrane is not due to platelet aggregates in the membrane.     

Immune Adherence of Platelets to Sensitized Leukocytes: a Model of Hyperacute Rejection

Specifically hypersensitized recipients of canine renal allotransplants demonstrate an accumulation of host platelets in the transplanted kidney within minutes following implantation. Transplant function is ra,idly lost. The present work describes an in vitro model of this hyperacute rejection mechanism. Renal donor leukocytes sensitized with host serum are incubated with donor platelets. Within a few minutes the platelets adhere to the sensitized leukocytes to form rosettes. Serum complement is required for platelet rosette formation. Platelets act as indicators in this reaction and they may be obtained from any dog. Donor platelets were used to avoid the transfer of specific antibody to the test mixture. The in vitro model supports the view that hyperacute rejection in the dog is a specific example of the immune adherence reaction in which platelets bind to antigen-antibody-complement complexes.     

Further characterization of the slow muscarinic responses inXenopus oocytes

In immature follicular ocytes of the frogXenopus laevis, application of muscarinic agonists evokes a complex response consisting of a fast and a slow Cl currents (the dominant responses), Cl current fluctuations, and a less prominent slow K current. The characteristics of the slow ACh-evoked potassium current were studied using the two-electrode voltage clamp method, and compared to those of the ACh-evoked Cl currents. In experiments designed to study the K current response separately, without the interference of ACh-evoked Cl currents, the holding potential was set close or equal to Cl equilibrium potential (measured as the reversal potential of the ACh-evoked Cl current). The Cl current responses were studied in cells that had negligible K current response. The dose-response curve of the potassium response followed classical Michaelis-Menten kinetics. The dose-response characteristics of the slow ACh-evoked Cl current displayed a positive cooperativity of at least 3. In spite of this difference, kinetic analysis revealed that these two responses, as well as the fast Cl current response that was characterized earlier (Dascal and Landau 1982), had almost identical apparent equilibrium dissociation constants (0.29–0.39 M), suggesting involvement of a single receptor class. Both K and Cl currents were reduced (to 32–56% of control) by millimolar concentrations of phosphodiesterase (PDE) inhibitors, theophylline and isobutylmethylxanthine. Elevation of extracellular Ca concentration from 1 to 10 mM doubled the K current; depletion of external Ca caused a partial inhibition of this response. The K current was potentiated by 0.1 M 4-phorbol 12,13-dibutyrate (PDBu). Ca-dependence of the ACh-evoked K current resembles that of ACh-evoked Cl currents, described earlier, and suggests mediation by a similar mechanism, i.e. mobilization of Ca from intracellular stores. On the other hand, most of the features described here are in a sharp contrast to those reported for adenosine-evoked, cAMP-mediated slow K current. Thus, we suggest that purinergic and muscarinic receptors inXenopus follicular oocytes are coupled to potassium channels through different molecular mechanisms.Abbreviations ACh acetylcholine - cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5-monophosphate - EGTA ethylenediaminetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-hydroxypropanesulphonic acid - IBMX 3-isobutyl-1-methylxanthine - IP₃ inositol 1,4,5-trisphosphate - PDBu 4-phorbol 12,13-dibutyrate - PDE phosphodiesterase     

Sialic acid and the surface charge of delayed rectifier potassium channels

We used the whole-cell configuration of the patch-clamp technique and cultured ventricular myocytes from 7-day embryonic chicks to test the hypothesis that sialic acid residues (NANA) constitute the negative surface charge associated with delayed rectifier potassium channels. Delayed rectifier current (iK) was elicited at potentials between -40 and +60 mV. The existence of negative fixed charges close to the "gating sensor" was confirmed by a 6.8-mV negative shift of the half-activation potential (V1/2) following a 10-fold reduction of divalent cations and a 22.6-mV position shift following the addition of 10 mM NiCl2. An 8.4-mV increase in the Boltzmann equation slope factor (k) in the former experiment and a 5.5-mV decline in the latter suggested that the surface charge is not uniformly distributed. We used a high performance liquid chromatography procedure to detect freed sarcolemmal NANA and found that 71-88% was released by neuraminidase (0.2-2.0 U/ml) during 1-h treatments. Such treatments had no significant effect upon the amplitudes of iK or V1/2. On the other hand, k was increased significantly by the enzyme (2.0 U/ml), but only when Ca2+ was present. Finally, 1-h pre-treatments with neuraminidase (2.0 U/ml) had no effect on the positive shift of V1/2 induced by Ni2+. We conclude that although sarcolemmal NANA may bind Ca2+, it does not constitute the surface charge of delayed rectifier potassium channels.     

Temporal Bone Histopathology in Connexin 26–Related Hearing Loss

Objective: Mutations in GJB2, a gene that encodes a gap junction protein, Connexin 26 (Cx26), are responsible for approximately one third of sporadic severe‐to‐profound or profound congenital deafness and half of severe‐to‐profound or profound autosomal recessive nonsyndromic hearing loss (ARNSHL). Mouse mutants homozygous for knockouts of this gene are nonviable, precluding histopathologic studies of the associated inner ear pathology in this animal model. Therefore, we studied archival temporal bone sections to identify temporal bone donors with Cx26‐related deafness. Study Design: Temporal bone donors with a history of congenital severe‐to‐profound or profound deafness were identified in the registry of the Temporal Bone Library at the University of Iowa. Histological findings were interpreted in a blinded fashion. DNA extracted from two celloidin‐embedded mid‐modiolar sections from each temporal bone was screened for the 35delG Cx26 mutation. The entire coding region of Cx26 was screened for other deafness‐causing mutations if the 35delG mutation was detected. Results: Of five temporal bone donors with congenital severe‐to‐profound deafness, one donor was found to have Cx26‐related deafness. This individual was a Cx26 compound heterozygote, carrying the 35delG mutation and a noncomplementary Cx26 missense mutation on the opposing allele. Microscopic evaluation of this temporal bone showed no neural degeneration, a good population of spiral ganglion cells, near‐total degeneration of hair cells in the organ of Corti, a detached and rolled‐up tectorial membrane, agenesis of the stria vascularis, and a large cyst in the scala media in the region of the stria vascularis. Conclusion: This study is the first to report the temporal bone histopathology associated with Cx26‐related deafness. Preservation of neurons in the spiral ganglion suggests that long‐term successful habilitation with cochlear implants may be possible in persons with severe‐to‐profound or profound Cx26‐related deafness.     

KINET: a social marketing programme of treated nets and net treatment for malaria control in Tanzania, with evaluation of child health and long-term survival.

We present a large-scale social marketing programme of insecticide-treated nets in 2 rural districts in southwestern Tanzania (population 350,000) and describe how the long-term child health and survival impact will be assessed. Formative and market research were conducted in order to understand community perceptions, knowledge, attitudes and practice with respect to the products to be socially marketed. We identified Zuia Mbu (Kiswahili for 'prevent mosquitoes') as a suitable brand name for both treated nets and single-dose insecticide treatment sachets. A mix of public and private sales outlets is used for distribution. In the first stage of a stepped introduction 31 net agents were appointed and trained in 18 villages: 15 were shop owners, 14 were village leaders, 1 was a parish priest and 1 a health worker. For net treatment 37 young people were appointed in the same villages and trained as agents. Further institutions in both districts such as hospitals, development projects and employers were also involved in distribution. Promotion for both products was intense and used a variety of channels. A total of 22,410 nets and 8072 treatments were sold during the first year: 18 months after launching, 46% of 312 families with children aged under 5 years reported that their children were sleeping under treated nets. A strong evaluation component in over 50,000 people allows assessment of the long-term effects of insecticide-treated nets on child health and survival, anaemia in pregnancy, and the costs of the intervention. This evaluation is based on cross-sectional surveys, and case-control and cohort studies.     

Progressive amplification and overexpression of the eukaryotic initiation factor 4E gene in different zones of head and neck cancers.

PURPOSE: Eukaryotic initiation factor 4E (eIF4E) binds to mRNA as the initial rate-limiting step in protein synthesis. Amplification and overexpression of the eIF4E gene has been associated with malignant transformation. The objectives of this study were to 1) quantify the eIF4E gene in head and neck squamous cell carcinoma (HNSCC) specimens, 2) quantify eIF4E protein elevation and examine its association with eIF4E gene amplification, and 3) determine whether there is progression in eIF4E gene amplification and protein overexpression in the tumor-free resection margin, the transition zone, and the tumor core of HNSCC specimens. MATERIALS AND METHODS: Eighteen HNSCC specimens were divided into three zones: 1) tumor core; 2) transition zone; and 3) "tumor-free" margin. Competitive polymerase chain reaction was performed to determine eIF4E gene copy number. eIF4E protein expression was quantified using Western blot analysis. RESULTS: All 18 HNSCC specimens tested had significant eIF4E gene amplification (4.3+/-1.2; P < .05). In contrast, none of the 10 benign specimens from noncancer patients had any eIF4E gene amplification (1.1+/-0.5). In the 12 HNSCC specimens examined for the three zones, the tumor core and transition zone showed eIF4E gene amplification (5.2+/-1.1 and 3.5+/-0.9, respectively) compared with the "tumor-free" margin (2.1+/-1.1; P < .05). The tumor core and transition zone showed significant efF4E protein elevation (15.5+/-9.3, 4.4+/-4.6, respectively) compared with the "tumor-free" margin (0.9+/-0.5, P < .05). CONCLUSIONS: The eIF4E gene is amplified and overexpressed in HNSCC. Amplification and elevation of eIF4E were highest in the tumor core, intermediate in the transition zone, and lowest in the tumor-free margin. There appears to be progression of eIF4E gene amplification and overexpression from the "tumor-free" margin to the tumor core.     

Profound hypoxemia during treatment of low cardiac output after cardiopulmonary bypass

PURPOSE: To illustrate the multiple causes of hypoxemia to be considered following cardiopulmonary bypass and how therapy given to improve oxygen delivery may have contributed to a decrease in arterial oxygen saturation to life-threatening levels. CLINICAL FEATURES: A 61 yr old man with severe mitral regurgitation and chronic obstructive lung disease underwent surgery for mitral valve repair. A pulmonary artery catheter with the capacity to measure cardiac output and mixed venous oxygen saturation (SvO2) continuously was used. Two unsuccessful attempts were made to repair the valve which was finally replaced, requiring cardiopulmonary bypass of 317 min. Dobutamine 5 micrograms.kg-1.min-1 and sodium nitroprusside 1 microgram.kg-1.min-1 were used to increase cardiac output. Soon after, the SvO2 decreased progressively from 55 to 39%. The patient became cyanotic with a PaO2 of 39 mmHg. Sodium nitroprusside was stopped and amrinone 100 mg bolus followed by 10 micrograms.kg-1.min-1 was given in addition to adding PEEP to the ventilation. With these measures PaO2 could be maintained of safe levels but PEEP and high inspired oxygen concentrations were needed postoperatively until the trachea could be extubated on the third postoperative day. CONCLUSION: The profound hypoxemia in this case was likely due to a combination of intra- and extrapulmonary shunt, both augmented by sodium nitroprusside. The desaturation of mixed venous blood amplified the effect of these shunts in decreasing arterial oxygen saturation. The interaction of these factors are analyzed in this report.     

'Brown bag' medication reviews as a means of optimizing patients' use of medication and of identifying potential clinical problems.

BACKGROUND: 'Brown bag' medication reviews carried out by community pharmacists collaborating with GPs have become established, in the USA and elsewhere, as an effective means of helping primary care patients to derive maximum benefit from their medicines, of identifying medication-related problems and of reducing wastage of medicines. OBJECTIVE: We aimed to determine whether 'brown bag' medication review could be used successfully in the UK, and particularly whether it represents an efficient and potentially cost-effective means of identifying medication problems. METHOD: 'Brown bag' medication reviews were carried out on 205 volunteer patients in 23 pharmacies in south-east London. Pharmacists' interventions to improve patients' knowledge and usage of their medicines were analysed. Potential clinical problems identified by pharmacists were analysed in order to identify the drug groups most likely to cause problems. RESULTS: Interventions were made in 87% of reviews; interventions to improve patients' knowledge of the purpose and correct usage of their drugs were made in 65% of reviews. In 12% of reviews, problems were identified that could potentially result in a hospital admission, and the potential for an improved outcome for the patient if drug therapy was changed was identified in a further 34% of cases. Beta-blockers, NSAIDs and verapamil were identified as being associated with potential problems of the highest clinical significance. Patients taking psychoactive medication were at greatest risk of a medication-related problem from any cause. CONCLUSION: Pharmacists could contribute to patients' welfare and reduce health care costs by carrying out 'brown bag' medication reviews on behalf of GPs.     

The effect of age and pre-light melatonin concentration on the melatonin sensitivity to dim light.

The hormone melatonin is secreted at night from the pineal gland, with light being a potent inhibitor of its secretion. Age related decreases in plasma melatonin concentrations have indicated that this may be related to pineal calcification with aging. Recently, it was shown that the melatonin sensitivity to light may be a biological marker of bipolar disorder. However, on average, patients were older than the control group in most studies, and it is not known if age has an effect on the melatonin suppression by light. To test this hypothesis, the present study investigated the effect of age on the melatonin sensitivity to dim light (200 lux). Participants were grouped into three age groups. On the testing night, they were placed in a dark room from 21.00 h to 02.30 h. Light exposure was for an hour from midnight to 01.00 h. Blood samples were collected at regular intervals for measurement of plasma melatonin. No significant differences were found in the percentage suppression of melatonin within the age groups defined in the present study (P > 0.5). No correlation was also found between age and percentage suppression of melatonin (r2 = 0.007; P > 0.1). Our results suggest that the melatonin suppression by light (200 lux) is not affected by age.