ATP-Evoked Intracellular Ca²âº Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea.

Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca(2+) signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca(2+)]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca(2+) transients in all three cell types, showing desensitization. Inhibiting the Ca(2+) signaling of the ionotropic P2X (omission of extracellular Ca(2+)) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca(2+) stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca(2+)-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca(2+) leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca(2+) signaling in these cells. Differences in Ca(2+) homeostasis and ATP-induced Ca(2+) signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology.

Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure.

Antenatal Bartter syndrome (aBS) comprises a heterogeneous group of autosomal recessive salt-losing nephropathies. Identification of three genes that code for renal transporters and channels as responsible for aBS has resulted in new insights into renal salt handling, diuretic action and blood-pressure regulation. A gene locus of a fourth variant of aBS called BSND, which in contrast to the other forms is associated with sensorineural deafness (SND) and renal failure, has been mapped to chromosome 1p. We report here the identification by positional cloning, in a region not covered by the human genome sequencing projects, of a new gene, BSND, as the cause of BSND. We examined ten families with BSND and detected seven different mutations in BSND that probably result in loss of function. In accordance with the phenotype, BSND is expressed in the thin limb and the thick ascending limb of the loop of Henle in the kidney and in the dark cells of the inner ear. The gene encodes a hitherto unknown protein with two putative transmembrane alpha-helices and thus might function as a regulator for ion-transport proteins involved in aBS, or else as a new transporter or channel itself.

The importance of different immunosuppressive regimens in the development of posttransplant diabetes mellitus.

Solid-organ transplantation is the optimal long-term treatment for most patients with end-stage organ failure. After solid-organ transplantation, short-term graft survival significantly improved (1). However, due to chronic allograft nephropathy and death with functioning graft, long-term survival has not prolonged remarkably (2). Posttransplant immunosuppressive medications consist of one of the calcineurin inhibitors in combination with mycophenolate mofetil (MMF) or azathioprine (Aza) and steroids. All of them have different adverse effects, among which posttransplant diabetes mellitus (PTDM) is an independent risk factor for cardiovascular (CV) events and infections causing the death of many transplant patients and it may directly contribute to graft failure (3). According to the criteria of the American Diabetes Association (4), diabetes mellitus (DM) is defined by symptoms of diabetes (polyuria and polydipsia and weight loss) plus casual plasma glucose concentration ≥ 11.1 mmol/L or fasting plasma glucose (FPG) ≥ 7.0 mmol/L or 2-h plasma glucose level ≥ 11.1 mmol/L following oral glucose tolerance test (OGTT). This metabolic disorder occurring as a complication of organ transplantation has been recognized for many years. PTDM, which is a combination of decreased insulin secretion and increased insulin resistance, develops in 4.9/15.9% of liver transplant patients, in 4.7/11.5% of kidney recipients, and in 15/17.5% of heart and lung transplants [cyclosporine A (CyA)/tacrolimus (Tac)-based regimen, respectively] (5). Risk factors of PTDM can be divided into non-modifiable and modifiable ones (6), among which the most prominent is the immunosuppressive therapy being responsible for 74% of PTDM development (7). Emphasizing the importance of the PTDM, numerous studies have determined the long-term outcome. On the basis of these studies, graft and patient survival is tendentiously (8) or significantly (9, 10) decreased for those developing PTDM.

[The German keratomycosis registry : Initial results of a multicenter survey]. / Das Deutsche Pilz-Keratitis-Register : Erste Ergebnisse einer multizentrischen Erhebung.

BACKGROUND AND PURPOSE: Mycotic keratitis is a serious but relatively rare disease. No targeted data collection in Germany existed until the foundation of the German Pilz-Keratitis Register in 2015. PATIENTS AND METHODS: The inclusion of retrospective and prospective patients was carried out. INCLUSION CRITERIA: diagnosis confirmed by the polymerase chain reaction (PCR), culture, histology or confocal microscopy (IVCM). Collected parameters: date of symptom onset, date and method of diagnosis, risk factors, visual acuity and findings at admission and at follow-up, conservative and surgical treatment. RESULTS: By January 2018, a total of 102 eyes from the years 2000-2017 were reported from 16 centers (64.3% female, mean age 52 years, range 18-95 years). The initial diagnosis was made correctly in only 20.6% of cases. The mean time to correct diagnosis was 31.7 ±â€¯46.9 (0-296) days. The diagnosis was confirmed in cultures in 74.5%, histologically in 30.4%, by PCR in 38.2% and IVCM in 27.4%. Fungal species identified were: 36.7% Fusarium spp., 35.8% Candida spp., 6.4% Aspergillus spp. and 21.1% other. The most important risk factor was the use of contact lenses. The most commonly used antifungal agent was voriconazole (64.7%) followed by amphotericin B (37.2%). Penetrating keratoplasty was performed in 65.7% of the cases and 8.8% of the affected eyes had to be enucleated. The visual acuity of the entire study population increased from the initial 0.16 ±â€¯0.25 (0.001-1.0) decimal to 0.28 ±â€¯0.34 (0-1.0) decimal. CONCLUSION: The correct diagnosis of fungal keratitis is often significantly delayed. The treatment can be very difficult and keratoplasty is often necessary. In order to gain a better understanding of this disease, to recognize previously unknown risk factors and, if necessary, a change in the spectrum of pathogens and to identify approaches to treatment optimization, the fungal keratitis registry will be continued.

The role of N-methyl-D-aspartate receptors and nitric oxide in cochlear dopamine release.

Dopamine (DA) released from lateral olivocochlear (LOC) terminals may have a neuroprotective effect in the cochlea. To explore the role of N-methyl-d-aspartate (NMDA) receptors and nitric oxide (NO) in the modulation of a cochlear DA release, we measured the release of [3H]DA from isolated mouse cochlea in response to the application of NMDA. NMDA at 100 muM significantly increased the electrical-field stimulation-evoked and resting release of DA from the cochlea. The NO donor sodium nitroprusside enhanced the basal outflow of DA but failed to influence the evoked release. The administration of the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) alone was ineffective, but it significantly inhibited the initial phase of the NMDA-induced elevation of DA outflow, which suggested the role of NO in the NMDA-induced DA release. The DA uptake inhibitor nomifensine increased the electrically evoked release of DA. Nomifensine failed to change the effect of NMDA on the resting or electrically-evoked DA release, which suggested that the uptake mechanism does not play a role in NMDA-evoked and NO-mediated DA release. In summary, we provide evidence that NO can modulate the release of DA from the cochlea following NMDA receptor activation, but does not affect the uptake of DA.

Anti-cyclic citrullinated peptide antibody isotypes in rheumatoid arthritis: association with disease duration, rheumatoid factor production and the presence of shared epitope.

OBJECTIVE: Anti-cyclic citrullinated peptide (anti-CCP) antibodies of IgG isotype are specific diagnostic markers of rheumatoid arthritis (RA). Recent evidence also points to their direct involvement in the pathophysiology. Little information is available, however, regarding the isotype distribution of anti-CCP antibodies and the characteristics of IgA and IgM anti-CCP. METHODS: IgG, IgA and IgM anti-CCP2 and rheumatoid factor (RF) levels were measured in the sera of 119 RA patients and 118 controls, including patients with other rheumatic diseases and healthy subjects. We analyzed the diagnostic performance of IgA and IgM anti-CCP2 antibodies and their relationship with IgG anti-CCP2, RFs, disease duration and the presence of HLA-DRB1 shared epitope (SE) alleles. RESULTS: Patients with RA had significantly higher serum IgA and IgM anti-CCP2 antibody levels than healthy subjects and patients with other rheumatic diseases (p<0.0001). IgG, IgA and IgM anti-CCP2 antibodies were present in 74.8%, 52.9% and 44.5% of RA patients, and their diagnostic specificity was 95.8%, 95.8% and 91.6%, respectively. The presence of anti-CCP2 antibodies was significantly associated with SE alleles (p=0.03). The frequency of IgM anti-CCP2 positivity was lower in longstanding disease compared to early RA (p=0.03). CONCLUSION: IgA and IgM anti-CCP2 antibodies are present in RA patients, and they are similarly specific for RA as IgG anti-CCP2. The higher frequency of IgM anti-CCP2 antibodies in early RA suggests that they are mostly generated during the first phase of immune response; nonetheless, their production seems to be sustained in some patients. Further analysis of IgM and IgA anti-CCP2 antibodies may provide insights into the pathogenesis of RA.

Post-transplant diabetes mellitus in children following renal transplantation.

PTDM plays a role in chronic allograft nephropathy and decreases graft and patient survival. Considering the serious outcome of chronic hyperglycemia, the importance of early recognition and the few data in children, in this retrospective analysis we studied the characteristics and risk factors of PTDM in 45 pediatric renal transplant recipients receiving Tac or CyA-based immunosuppression. Fasting blood sampling and OGTT were performed. PTDM has been developed in six patients (13%), while seven children (16%) had IGT, with the overall incidence of a glucose metabolic disorder of 29% in pediatric renal transplants. Patients in the PTDM + IGT group were younger and had higher systolic blood pressure and serum triglyceride level than children with normal glucose tolerance. Multivariate analysis identified Tac treatment, Tac trough level, steroid pulse therapy and family history of diabetes to be associated with the onset of PTDM. In pediatric renal transplants, OGTT and frequent assessment of blood glucose levels might be essential not only in the post-transplant management, but also prior to transplantation, particularly with family history of diabetes. Careful monitoring and modified protocols help to minimize the side effects of Tac and corticosteroids.

Cigarette smoke-induced alterations in endothelial nitric oxide synthase phosphorylation: role of protein kinase C.

Endothelial nitric oxide synthase (eNOS) is regulated by phosphorylation of Ser(1177) and Thr(495), which affects NO bioavailability. Cigarette smoke disturbs the eNOS-cGMP-NO pathway and causes decreased NO production. Here the authors investigated the acute effects of cigarette smoke on eNOS phosphorylation, focusing on protein kinases (PKs). Endothelial cell culture was concentration- and time-dependently treated first with cigarette smoke buffer (CSB), then with reduced glutathione (GSH) or various PK inhibitors (H-89, LY-294002, Ro-318425, and ruboxistaurin). eNOS, phospho-Ser(1177)-eNOS, phospho-Thr(495)-eNOS, Akt(PKB), and phospho-Akt protein levels were determined by Western blot. CSB increased the phosphorylation of eNOS at Ser(1177) and more at Thr(495) in a concentration- and time-dependent manner (p < .01, p < .05 versus control, respectively) and resulted in the dissociation of the active dimeric form of eNOS (p < .05). GSH decreased the phosphorylation of eNOS at both sites (p < .05 versus CSB without GSH) and prevented the decrease of dimer eNOS level. CSB treatment also decreased the level of phospho-Ser(473)-Akt (p < .05 versus control). Inhibition of PKA by H-89 did not affect CSB-induced phosphorylation, whereas the PKB inhibitor LY-294002 enhanced it at Ser(1117). The PKC blockers Ro-318425 and ruboxistaurin augmented the CSB-induced phosphorylation at Ser(1177) but decreased phosphorylation at Thr(495) (p < .05 versus CSB). Cigarette smoke causes a disruption of the enzymatically active eNOS dimers and shifts the eNOS phosphorylation to an inhibitory state. Both effects might lead to reduced NO bioavailability. The shift of the eNOS phosphorylation pattern to an inhibitory state seems to be independent of the PKA and phosphoinositol 3-kinase (PI3-K)/Akt pathways, whereas PKC appears to play a key role.

The effect of the short wavelength ultraviolet radiation. An extension of biological dosimetry to the UV-C range.

Polycrystalline uracil thin layer can be used as biological dosimeter for assessing exposure to UV radiation. The dimerization and reversion efficiency of the ultraviolet radiation in the UV-B and the UV-C range were quantified on polycrystalline uracil thin layers irradiated with quasi-monochromatic radiation using interference filters of 10nm bandwidth. The dimer formation and monomerization (reversion) dose-effect relations were determined by optical spectroscopy. The decrease of the OD value of the uracil thin layer at 288 nm was taken as a measure of the dimer formation, while the increase of the OD of a completely irradiated (until reaching the saturation level) uracil layer was taken as the sign of the monomerization. The two processes in the UV-B and the UV-C range take place simultaneously, the individual characterization of the dimerization efficiency was performed from the initial slope of the dimerization dose-effect function and an action spectrum for dimerization was constructed in the UV-C range too. The reversion efficiency was found to be practically the same with all of the investigated wavelengths: 200 nm, 210 nm, 220 nm, 230 nm, 240 nm The possible biological relevance of the reversion of dimers are discussed.

Dietary practices in isovaleric acidemia: A European survey.

BACKGROUND: In Europe, dietary management of isovaleric acidemia (IVA) may vary widely. There is limited collective information about dietetic management. AIM: To describe European practice regarding the dietary management of IVA, prior to the availability of the E-IMD IVA guidelines (E-IMD 2014). METHODS: A cross-sectional questionnaire was sent to all European dietitians who were either members of the Society for the Study of Inborn Errors of Metabolism Dietitians Group (SSIEM-DG) or whom had responded to previous questionnaires on dietetic practice (n = 53). The questionnaire comprised 27 questions about the dietary management of IVA. RESULTS: Information on 140 patients with IVA from 39 centres was reported. 133 patients (38 centres) were given a protein restricted diet. Leucine-free amino acid supplements (LFAA) were routinely used to supplement protein intake in 58% of centres. The median total protein intake prescribed achieved the WHO/FAO/UNU [2007] safe levels of protein intake in all age groups. Centres that prescribed LFAA had lower natural protein intakes in most age groups except 1 to 10 y. In contrast, when centres were not using LFAA, the median natural protein intake met WHO/FAO/UNU [2007] safe levels of protein intake in all age groups. Enteral tube feeding was rarely prescribed. CONCLUSIONS: This survey demonstrates wide differences in dietary practice in the management of IVA across European centres. It provides unique dietary data collectively representing European practices in IVA which can be used as a foundation to compare dietary management changes as a consequence of the first E-IMD IVA guidelines availability.

Dietary practices in propionic acidemia: A European survey.

BACKGROUND: The definitive dietary management of propionic acidaemia (PA) is unknown although natural protein restriction with adequate energy provision is of key importance. AIM: To describe European dietary practices in the management of patients with PA prior to the publication of the European PA guidelines. METHODS: This was a cross-sectional survey consisting of 27 questions about the dietary practices in PA patients circulated to European IMD dietitians and health professionals in 2014. RESULTS: Information on protein restricted diets of 186 PA patients from 47 centres, representing 14 European countries was collected. Total protein intake [PA precursor-free L-amino acid supplements (PFAA) and natural protein] met WHO/FAO/UNU (2007) safe protein requirements for age in 36 centres (77%). PFAA were used to supplement natural protein intake in 81% (n = 38) of centres, providing a median of 44% (14-83%) of total protein requirement. Seventy-four per cent of patients were prescribed natural protein intakes below WHO/FAO/UNU (2007) safe levels in one or more of the following age groups: 0-6 m, 7-12 m, 1-10 y, 11-16 y and > 16 y. Sixty-three per cent (n = 117) of patients were tube fed (74% gastrostomy), but only 22% received nocturnal feeds. CONCLUSIONS: There was high use of PFAA with intakes of natural protein commonly below WHO/FAO/UNU (2007) safe levels. Optimal dietary management can only be determined by longitudinal, multi-centre, prospective case controlled studies. The metabolic instability of PA and small patient cohorts in each centre ensure that this is a challenging undertaking.

Photosensitized inactivation of T7 phage as surrogate of non-enveloped DNA viruses: efficiency and mechanism of action.

We investigated the efficiency and the mechanism of action of a tetraphenyl porphyrin derivative in its photoreaction with T7 phage as surrogate of non-enveloped DNA viruses. TPFP was able to sensitize the photoinactivation of T7 phage in spite of the lack of its binding to the nucleoprotein complex. The efficiency of TPFP photosensitization was limited by the aggregation and by the photobleaching of porphyrin molecules. Addition of sodium azide or 1,3-dimethyl-2-thiourea (DMTU) to the reaction mixture moderated T7 inactivation, however, neither of them inhibited T7 inactivation completely. This result suggests that both Type I and Type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated nucleoprotein complex. Polymerase chain reaction (PCR) also failed to demonstrate any DNA damage. Circular dichroism (CD) spectra of photosensitized nucleoprotein complex indicated changes in the secondary structure of both the DNA and proteins. We suggest that damages in the protein capsid and/or loosening of protein-DNA interaction can be responsible for the photodynamic inactivation of T7 phage. The alterations in DNA secondary structure might be the result of photochemical damage in phage capsid proteins.

Improving techniques for clonogenic assays.

A serum-free medium has been developed which supports colony formation by cells from several human tumor cell lines, one colon adenocarcinoma (WiDr) and four melanoma (Me43, Me85, MP6, MeIuso). This medium consists of a 1:1 mixture of an enriched Dulbecco's modified Eagle's medium (EMED) and a modified Ham's F-12 nutrient mixture (FMED) supplemented with 0.9% methylcellulose, 1% bovine serum albumin, 80 micrograms/ml human transferrin, 3 micrograms/ml insulin, 2.8 micrograms/ml linoleic acid, 2.6 micrograms/ml cholesterol, 20 microM ethanolamine, and trace elements. Colony formation by WiDr cells is linear with the numbers of cells plated, having a plating efficiency (PE) of 34%, as compared to 26% in serum-containing medium. Two of the melanoma cell lines. MP6 and MeIuso, exhibit linear relationships between colony numbers and cell concentration with PEs of 21% and 70% respectively. Colony formation by the other two melanoma cell lines appears to be nonlinear. This work represents a step toward standardizing culture conditions for human tumor clonogenic cell assays.

Validation of phage T7 biological dosimeter by quantitative polymerase chain reaction using short and long segments of phage T7 DNA.

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.

Assessment of the effects of various UV sources on inactivation and photoproduct induction in phage T7 dosimeter.

The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources. The BED is the inactivation rate of phage T7 expressed in HT7 units. The CPD and (6-4)PD were determined by lesion-specific monoclonal antibodies in an immunodot-blot assay. The various lamps induced these lesions at different rates; the relative induction ratios of CPD to (6-4)PD increased with increasing effective wavelength of irradiation source. The amount of total adducts per phage was compared to the BED of phage T7 dosimeter, representing the average number of UV lesions in phage. For UVC (200-280 nm radiation) and unfiltered TL01 the number of total adducts approximates the reading; however, UV sources having longer effective wavelengths produced fewer CPD and (6-4)PD. A possible explanation is that although the most relevant lesions by UVC are the CPD and (6-4)PD, at longer wavelengths other photoproducts can contribute to the lethal damage of phages. The results emphasize the need to study the biological effects of solar radiation because the lesions responsible for the lethal effect may be different from those produced by various UV sources.

Influence of phage proteins on formation of specific UV DNA photoproducts in phage T7.

Phage T7 can be used as a biological UV dosimeter. Its reading is proportional to the inactivation rate expressed in HT7 units. To understand the influence of phage proteins on the formation of DNA UV photoproducts, cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts ((6-4)PD) were determined in T7 DNA exposed to UV radiation under different conditions: intraphage T7 DNA, isolated T7 DNA and heated phage. To investigate the effects of various wavelengths, seven different UV sources have been used. The CPD and (6-4)PD were determined by lesion-specific antibodies in an immunodot-blot assay. Both photoproducts were HT7 dose-dependently produced in all three objects by every irradiation source in the biologically relevant UV dose range (1-10 HT7). The CPD to (6-4)PD ratios increased with the increasing effective wavelength of the irradiation source and were similar in intraphage T7 DNA, isolated DNA and heated phage with all irradiation sources. However, a significant decrease in the yield of both photoproducts was detected in isolated T7 DNA and in heated phage compared to intraphage DNA, the decrease was dependent on the irradiation source. Both photoproducts were affected the same way in isolated T7 DNA and heated phage, respectively. The yield of CPD and (6-4)PD was similar in B, C-like and A conformational states of isolated T7 DNA, indicating that the conformational switch in the DNA is not the decisive factor in photoproduct formation. The most likely explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in an increased rate of dimerization and (6-4)PD production of adjacent based in intraphage T7 DNA.

Photoinduced inactivation of T7 phage sensitized by symmetrically and asymmetrically substituted tetraphenyl porphyrin: comparison of efficiency and mechanism of action.

We investigated the efficiency and the mechanism of action of two--one symmetrically and one asymmetrically substituted--glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 phage as a model of nucleoprotein (NP) complexes. A correlation was found between the dark inactivation of T7 and the binding of porphyrins determined by fluorescence spectroscopy. Both types of porphyrin sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different. There was no correlation between the dark binding and the photosensitizing efficacy of the two derivatives. Inactivation was moderated by 1,3-diphenylisobenzofuran and 1,3-dimethyl-2-thiourea; however, neither of them inhibited T7 inactivation completely. This result suggests that both Type-I and Type-II reactions play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated NP complex. Polymerase chain reaction analysis of a 555 bp segment of gene 1 and a 3826 bp segment of genes 3 and 4 failed to demonstrate any DNA damage.

Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in hemolymph of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in hemolymph collected from live Dermacentor andersoni Stiles ticks. Hemolymph was collected from severed legs of male and female ticks exposed to A. marginale as either nymphs or adults. Heat treatment was found to be the optimum method of hemolymph preparation for PCR. Hemolymph samples were collected and pooled from adult ticks exposed as nymphs on days 0-10 of feeding on a susceptible calf. For male and female ticks exposed as adults, samples were collected as ticks fed 7 d on an infected calf, while being held 9 d between feedings, and during a second feeding of 10 d (or to repletion) when they transmitted the parasite. Hemolymph samples were collected from uninfected ticks at the same times to serve as controls. Anaplasma marginale DNA was amplified with primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'), which flank a 409-bp fragment of the A. marginale Florida isolate msp1 beta gene. Infected tick hemolymph was PCR-positive for A. marginale at all collection times, including unfed adults infected as nymphs and previously unexposed adults that fed on infected calves for only 1 d. The PCR-based assay of tick hemolymph proved to be a sensitive method for identification of infected ticks, potentially without killing them; it would be well suited for identification of laboratory- or field-infected ticks that could then be used for further studies. The primers used in this assay were also found specific when tested with species of 18 different genera, and universal for 7 A. marginale isolates from diverse geographical areas of the United States.

Analysis of X-ray-induced HPRT mutations in CHO cells: insertion and deletions.

Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TGR) Chinese hamster ovary (CHO) cell clones. Rapid screening of the clones by multiplex polymerase chain reaction (PCR) for the presence or absence of exons revealed that the causes of the mutant phenotype were total gene deletion (26/41), partial gene deletion (4/41), and an insertion (1/41). No alterations of exon number or sizes were apparent in 10 of the mutants. Southern blot analysis confirmed the deletion data and revealed an additional class of mutants that had a gene disruption but retained all hprt exons (2/41). Therefore, at least 80% of the ionizing radiation-induced mutations were due to mechanisms involving DNA breakage and rejoining. The distribution of deletion sizes suggests that the two DNA breaks required for a deletion are not independent events. A possible mechanism is presented. In addition, the DNA sequence of the insertion mutation was determined. The insertion (229 bp) is coupled with a deletion (31 bp). An imperfect inverted repeat with flanking hprt DNA was identified and may be involved in the insertion event.

Effect of propranolol on heart rate variability in patients with end-stage renal disease: a double-blind, placebo-controlled, randomized crossover pilot trial.

BACKGROUND: Low heart rate variability (HRV) is an independent risk factor of cardiac mortality in patients with end-stage renal disease (ESRD). It has been explained by uremic parasympathetic neuropathy. Sympathetic overactivity can also reduce HRV. Our aim was to determine whether there is vagal activity in ESRD patients that is masked by sympathetic activity. METHODS: The effect of propranolol on HRV was examined in 13 patients with ESRD, aged 20.1 +/- 7.6 years without diabetes. All patients were given intravenous propranolol (0.05 mg/kg) once and placebo once in a randomized, double-blind way, with an interval of 6.6 days (mean, range: 2-9). Propranolol was administered before hemodialysis treatment, after 40 minutes supine resting period. HRV was registered for 10 minutes, during supine, before and after the injection. Patients' HRV data were compared to that of 29 age-matched healthy controls. RESULTS: Initially, both high-(HFV) and low-frequency (LFV) bands of heart rate variability were lower in ESRD patients compared to controls (p < 0.001 for both). Propranolol resulted in a significant increase of HFV (propranolol: AlgHFV = 0.182 (0.027 - 0.337), placebo: deltalgHFV = -0.029 (-0.128 - +0.070); p = 0.032). Elevation of LFV was not significant. Six patients had an elevated plasma norepinephrine and/or epinephrine level. Plasma dopamine level was elevated in all but 1 patient (mean: 432 pmol/l, 95% CI: 320-543) and showed an inverse relationship with the increase of IgHFV secondary to propranolol (r = -0.66, p = 0.014). CONCLUSIONS: Low HFV of ESRD patients can be improved by beta-adrenergic blockade. It demonstrates that there is some vagal activity in ESRD that is masked by sympathetic activity. Therefore, altered sympathovagal balance of ESRD patients should be taken into consideration in the assessment of vagal uremic neuropathy.