Creating models for the prediction of colostrum quantity, quality, and immunoglobulin G yield in multiparous Jersey cows from performance in the previous lactation and environmental changes.

With multiparous Jersey cows, colostrum production seems to be variable. Due to this, we aimed to identify specific variables involved in colostrum production and quality. From 2021 to 2023, data from 28 US farms (415 multiparous Jersey cows) were used to investigate if colostrum yield, IgG concentration (g/L), and IgG yield (g) could be predicted by farm variables and transmitting abilities. With the data collected, multiple regression equations were developed to aid in predicting colostrum yield, IgG concentration, and IgG yield. Colostrum was weighed and sampled for IgG analysis. Dairy Herd Information (DHI), calving, diet, and management information data were compiled. Days below 5°C (D<), days above 23°C (D>), and days between 5 and 23°C (D) were recorded. We evaluated transmitting abilities for milk, fat, protein, and dollars; previous lactation milk yield, fat percent, fat yield, protein percent, protein yield, previous lactation somatic cell score, previous lactation days open, previous lactation days dry, previous lactation days in milk, and previous parity; and current lactation parity, days dry, and calving information, birth ordinal day, and latitude. Colostrum yield, IgG yield, and concentration had 1 added to correct for values = 0. After addition, values >0 were transformed to ln or log10. Nontransformed variables were also used to develop the model. Variance inflation factor analysis was conducted, followed by backward elimination. The log10 colostrum yield model (R2 = 0.55; ß in parentheses) included herd size (-0.0001), ordinal days (-0.001), ln ordinal days (0.07), latitude (-0.02), dry period length (0.004), D< (-0.005), D (-0.003), time to harvest (0.05), ln time to harvest (-0.35), IgG (-0.004), log10 IgG (0.46), feedings per day (0.06), ln pasture access (-0.13), and ln previous lactation days open (0.14). The model showed that previous lactation days open contributed the most toward increasing and latitude contributed the most toward decreasing colostrum yield. The IgG model (R2 = 0.21) included herd size (0.02), D> (0.38), ln time to harvest (-19.42), colostrum yield (-4.29), ln diet type (18.00), ln previous lactation fat percent (74.43), and previous parity (5.72). The model showed that previous lactation milkfat percent contributed the most toward increasing and time from parturition to colostrum harvest contributed the most toward decreasing colostrum IgG concentration. The log10 IgG yield model (R2 = 0.79) included ln ordinal days (0.03), time to harvest (-0.01), colostrum yield (-0.11), ln colostrum yield (1.20), ln pasture access (-0.09), ln previous lactation fat percent (0.53), and previous parity (0.02). The model showed that colostrum yield contributed the most toward increasing IgG yield, followed by previous lactation milkfat percentage. Pasture access contributed the most toward decreasing IgG yield, although the contribution was very small. These models were validated using 39 samples from 22 farms. Actual minus predicted colostrum yield and IgG concentration and yield were 0.89 kg, -21.10 g/L, and -65.15 g, respectively. These models indicate that dry period management and cow information can predict colostrum yield and IgG concentration and yield.

Perspective of dairy producers from California, Idaho, South Dakota, and Washington: Health and business implications of the COVID-19 pandemic during the second wave.

The aim of this study was to document California, Idaho, South Dakota, and Washington dairy producers' perspectives on the health and business implications of COVID-19 during the pandemic second wave. Dairy producers were reached by a 14-question anonymous mail survey during June and July of 2020. We obtained 226 responses (response rate: 9.3% CA, 8.6% ID, 31.4%, and 10.0% WA). Responses were grouped by state (CA: 48.7%, ID: 15.9%, SD: 21.7%, or WA: 13.7%) and dairy size [based on number of cows; small (<100): 14.1%, medium (100-499): 27.7%, medium-large (500-1,999): 33.2%, or large (≥2,000): 25.0%]. Survey responses were summarized, and multiple correspondence analysis was used to map responses and identify data clusters. At the time of the survey, some respondents suspected (3%) or had confirmed (9%) COVID-19 cases on their premises. Respondents were somewhat or very concerned about the health (75%) and business (92%) implications associated with COVID-19. Producers were concerned about the health of their families, employees, and employees' families; having to reduce production; workforce shortage; limited goods availability; and lack of services. Producers perceived that their employees were somewhat or very concerned (74%) with the pandemic. There was COVID-19 safety information or training provided (78%) or intended (4%) in English (22%), Spanish (23%), or both (55%). The focus of training was as follows: how to remain healthy at work (91%) and at home (60%), what to do if a worker gets sick (77%), and sick leave information (54%). Nevertheless, 18% of the respondents answered that training was not going to be provided. The following control measures implemented: providing hand sanitizer or disinfectant wipes (78%), instruction on frequent hand-washing (76%), social distancing (59%), prevention of employee gatherings (54%), providing and requiring the use of face masks (49%), and limiting on-farm visitors (44%). Multiple correspondence analysis showed that concern with health risk and business impact of the COVID-19 pandemic, as well as mitigation and response strategies implemented, varied based on respondent's dairy size and state. The greatest concerns were reported by producers from California and large and medium-large dairies, whereas the fewest concerns were reported by producers from small dairies. Results from this survey highlighted the health and business concerns of dairy producers from California, Idaho, South Dakota, and Washington during the COVID-19 pandemic and the mitigation efforts adopted. In conclusion, mitigation measures were adopted by most dairy producers but in various degrees. Dairy industry service providers and educators were secondary educational resources during the pandemic; thus, future efforts toward centralized access to dairy specific bilingual educational materials are suggested.

Stroke symptoms impact on mental and physical health: A Lebanese population based study.

BACKGROUND/OBJECTIVE: Stroke symptoms in the absence of diagnosed stroke are common worldwide and associated with stroke risk factors and great impact on the physical and mental health functioning. The aim of this study was to assess, at the national level, the association of stroke symptoms with mental and physical health. METHODS: Quality of life was assessed using physical and mental component summary scores (PCS and MCS) of the Short Form 12v2 Health Survey in the Lebanese population. We assessed the differences in the mean PCS and MCS scores among asymptomatic individuals with no stroke/transient ischemic attack (TIA) history (n=1167), symptomatic individuals with no stroke/TIA history (n=125) and those with stroke/TIA history (n=46). Psychometric properties of the Lebanese version of the SF- 12v2 were evaluated using principal component analysis. RESULTS: Symptomatic individuals had an average PCS scores of 2.31 (95%CI: 0.75-3.88) points lower and those with stroke/TIA history had 3.26 (95%CI: 1.01-5.51) points lower when compared with asymptomatic individuals with no stroke/TIA history. Similarly, MCS scores for symptomatic individuals were 2.58 (95%CI: 1.02-4.13) points lower and those with stroke/TIA history had 3.28 (95%CI: 1.06-5.50) points lower than asymptomatic individuals. CONCLUSION: Physical and mental health functioning declined among symptomatic individuals and those with stroke/TIA history. Thus, frequent monitoring for the early detection of stroke symptoms may be recommended.

Mechanisms of Drug Binding to Voltage-Gated Sodium Channels.

Voltage-gated sodium (Na+) channels are expressed in virtually all electrically excitable tissues and are essential for muscle contraction and the conduction of impulses within the peripheral and central nervous systems. Genetic disorders that disrupt the function of these channels produce an array of Na+ channelopathies resulting in neuronal impairment, chronic pain, neuromuscular pathologies, and cardiac arrhythmias. Because of their importance to the conduction of electrical signals, Na+ channels are the target of a wide variety of local anesthetic, antiarrhythmic, anticonvulsant, and antidepressant drugs. The voltage-gated family of Na+ channels is composed of α-subunits that encode for the voltage sensor domains and the Na+-selective permeation pore. In vivo, Na+ channel α-subunits are associated with one or more accessory ß-subunits (ß1-ß4) that regulate gating properties, trafficking, and cell-surface expression of the channels. The permeation pore of Na+ channels is divided in two parts: the outer mouth of the pore is the site of the ion selectivity filter, while the inner cytoplasmic pore serves as the channel activation gate. The cytoplasmic lining of the permeation pore is formed by the S6 segments that include highly conserved aromatic amino acids important for drug binding. These residues are believed to undergo voltage-dependent conformational changes that alter drug binding as the channels cycle through the closed, open, and inactivated states. The purpose of this chapter is to broadly review the mechanisms of Na+ channel gating and the models used to describe drug binding and Na+ channel inhibition.

Perspectives of dairy employees at the beginning of the COVID-19 pandemic: A survey of health risks and educational needs.

Our objective was to understand dairy employees' perceptions and educational needs at the onset of the COVID-19 pandemic. A bilingual (English and Spanish), anonymous survey targeted at dairy employees was circulated nationwide via university and allied industry media outlets. Responses (n = 63) from 11 states were received (May-Sep. 2020). Respondents worked in herds ranging from 50 to 40,000 animals in size. Dairy managers (33%) responded mostly to the English survey (52%), whereas entry-level workers (67%) chose the Spanish format (76%). Survey results highlighted different perspectives, educational needs, and preferred sources of information between English- and Spanish-speaking dairy workers. Overall, 83% of the respondents were somewhat concerned or very concerned about the COVID-19 pandemic. Respondents (51%) indicated that their main concern was "to bring the virus from work to home and make my family sick." Most dairy employees (83%) perceived that their employers were somewhat or very concerned about the pandemic. Respondents (65%) indicated that COVID-19 informative training was provided at the workplace, but training was more frequently undertaken among dairy managers (86%) than entry-level workers (53%). Most trainings (72%) were limited to posters on walls. The preferred means of information delivery was through in-person meetings at work (35%), with YouTube (29%) and on-demand videos (27%) as second and third options. The main source of information regarding the pandemic was social media (52%). Frequent handwashing (81%), limiting on-farm visits (70%), limiting agglomeration in break rooms (65%), hand sanitizer use (60%), and social distancing (60%) were the most common safety measures implemented at the workplace among the options given to respondents. Few respondents (38%) indicated that face-covering was required at work. Successful emergency plans on dairies should consider the outreach needs and preferences of dairy workers.

Molecular analysis of imipenem-resistant Acinetobacter baumannii isolated from US service members wounded in Iraq, 2003-2008.

Global dissemination of imipenem-resistant (IR) clones of Acinetobacter baumannii - A. calcoaceticus complex (ABC) have been frequently reported but the molecular epidemiological features of IR-ABC in military treatment facilities (MTFs) have not been described. We characterized 46 IR-ABC strains from a dataset of 298 ABC isolates collected from US service members hospitalized in different US MTFs domestically and overseas during 2003-2008. All IR strains carried the bla(OXA-51) gene and 40 also carried bla(OXA-23) on plasmids and/or chromosome; one carried bla(OXA-58) and four contained ISAbal located upstream of bla(OXA-51). Strains tended to cluster by pulsed-field gel electrophoresis profiles in time and location. Strains from two major clusters were identified as international clone I by multilocus sequence typing.

Effects of dietary betaine on milk yield and milk composition of mid-lactation Holstein dairy cows.

Betaine, naturally found in plants and an oxidative product of choline, is converted to acetate in the rumen, which may be used for milk fat synthesis. The objective of this study was to determine the effect of supplemental dietary betaine on milk yield and milk composition. Eighteen Holstein dairy cows (126±5 d in milk; mean ± SD) were randomly assigned to a sequence of treatments of rumen-unprotected betaine at 0, 25, 50, and 100 g/d added to a standard lactation ration in a 4×4 Latin square design. Animals were fed individually with feed intake and milk yield recorded daily. Body condition score and body weight were recorded on the last day of each period that lasted 16 d, with milk sampled on the last 2 d of each period. Milk composition was determined by a Dairy Herd Improvement Association laboratory and milk fatty acids were determined by gas chromatography. Data collected over the last 2 to 3 d were analyzed using the MIXED procedure in SAS (SAS Institute Inc., Cary, NC). Milk yield (mean ± SEM) was increased by betaine when fed at 100g/d (22.4, 22.5, 22.8, 24.1±1.19 kg/d for 0, 25, 50, and 100g of betaine/d, respectively). No effect of dietary betaine was detected on dry matter intake, feed efficiency, body weight, or body condition score. Percentages of milk fat, lactose, solids-not-fat, and somatic cell count were not altered; however, protein concentration was decreased by betaine supplementation as compared with the control (3.35, 3.28, 3.27, and 3.28±0.07% for 0, 25, 50, and 100 g of betaine/d, respectively). Daily yields of milk protein, fat, lactose, energy-corrected milk, and 3.5% fat-corrected milk did not differ with betaine supplementation. Overall, inclusion of dietary betaine at 100 g/d increased milk yield, whereas all levels of betaine supplementation decreased milk protein percent and slightly altered milk fatty acid profile. Further studies are needed to determine the ruminal fermentation characteristics and the optimum rate of supplemental betaine for dairy cows.

Methicillin-resistant Staphylococcus aureus infection in combat support hospitals in three regions of Iraq.

SUMMARYStaphylococcus aureus is a leading cause of infections in deployed service members. Based on a molecular epidemiological study of 182 MRSA isolates from patients in three U.S. Army combat support hospitals in separate regions in Iraq, USA300 clone was the most predominant (80%) pulsotype. This finding suggested that strain carriage from the home country by military personnel is epidemiologically more important than local acquisition.

Changes in action potentials and intracellular ionic homeostasis in a ventricular cell model related to a persistent sodium current in SCN5A mutations underlying LQT3.

In LQT3 patients, SCN5A mutations induce ultraslow inactivation of a small fraction of the hNav1.5 current, i.e. persistent Na+ current (IpNa). We explored the time course of effects of such a change on the intracellular ionic homeostasis in a model of guinea-pig cardiac ventricular cell [Pasek, M., Simurda, J., Orchard, C.H., Christé, G., 2007b. A model of the guinea-pig ventricular cardiomyocyte incorporating a transverse-axial tubular system. Prog. Biophys. Mol. Biol., this issue]. Sudden addition of IpNa prevented action potential (AP) repolarization when its conductance (gpNa) exceeded 0.12% of the maximal conductance of fast INa (gNa). With gpNa at 0.1% gNa, the AP duration at 90% repolarization (APD90) was initially lengthened to 2.6-fold that in control. Under regular stimulation at 1 Hz it shortened progressively to 1.37-fold control APD90, and intracellular [Na+]i increased by 6% with a time constant of 106 s. Further increasing gpNa to 0.2% gNa caused an immediate increase in APD90 to 5.7-fold that in control, which decreased to 2.2-fold that in control in 30s stimulation at 1 Hz. At this time diastolic [Na+]i and [Ca2+]i were, respectively, 34% and 52% higher than in control and spontaneous erratic SR Ca release occurred. In the presence of IpNa causing 46% lengthening of APD90, the model cell displayed arrhythmogenic behaviour when external [K+] was lowered to 5 mM from an initial value at 5.4 mM. By contrast, when K+ currents IKr and IKs were lowered in the model cell to produce the same lengthening of APD90, no proarrhythmic behaviour was observed, even when external [K+] was lowered to 2.5 mM.

Assessment of management and basic beef quality assurance practices on Idaho dairies.

In 2004 a mail-in survey was conducted to establish a baseline level of awareness and knowledge related to dairy beef quality assurance (BQA) issues in Idaho. A 30-question survey was mailed to every (n = 736) registered Idaho dairy. Two-hundred seventy-three (37%) dairies participated and were categorized as small (n <201 cows; 53.5%), medium-sized (n = 201 to 1,000 cows; 27.1%) or large (n >1,000 cows; 19.4%). The majority of respondents were dairy owners (83%). Eighty-nine percent of respondents indicated they followed BQA recommendations for animal care. The neck region in cows was used by 68% of respondents for i.m. injections and by 80% for s.c. injections. In calves, the values were 61 and 78%, respectively. Seventy-four percent of respondents indicated they had been trained for injections. Training methods cited included veterinarians (19.8%), dairy owners (16.8%), experience (9.9%), and BQA events or schools (4.5%). The importance of BQA in the dairy industry was rated 2.6 on a 5-point scale (0 = low; 4 = high). Participants rated the effect of dairy animals on the beef industry at 2.5. Plastic ear tags were the preferred method of animal identification, with 100% of large dairies, 97.3% of medium-sized dairies, and 84% of small dairies citing their use. Less than 10% used electronic identification for their animals. Almost half (48%) of large and medium-sized (49%) dairies and 32% of small dairies supported a national animal identification program. A mandatory identification program was supported by 41, 69, and 59% for small, medium-sized, and large dairies, respectively. The percentage of dairies keeping records was similar between small (93%), medium-sized (99%), and large (100%) dairies. Most small dairies (58%) used some form of paper records, whereas most medium-sized (85%) and large (100%) dairies used computers for record keeping. The preferred method to market cull cows by Idaho dairies was the auction market (64%), followed by order buyers (17%), direct to the packer (17%), private treaty sales (16%), and forward contracts (1%). To market calves, dairies used private treaty sales (52%), auction markets (42%), order buyers (14%), and forward contracts (1%). The results of this study will be used by University of Idaho Extension faculty in the design, development, and delivery of dairy BQA program information and materials.

First Lebanese Antibiotic Awareness Week campaign: knowledge, attitudes and practices towards antibiotics.

Antibiotic resistance (ABR) is a major global health threat that increases the risk of treatment failure and increases medical costs. One of the most common factors contributing to the spread of ABR is self-medication. The public, as well as workers in clinical and veterinary sectors, commit false practices towards appropriate antibiotic use, favouring the spread of resistance. As such, the first Lebanese Antibiotic Awareness Week campaign was initiated with a human-centred and interactive approach. The data showed a strikingly low level of antibiotic awareness. Cooperation between relevant stakeholders, policy-makers and health actors is crucial to control and overcome the problem of ABR.

Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation.

Mutations in the adult human skeletal muscle Na+ channel alpha subunit cause the disease paramyotonia congenita. Two paramyotonia congenita mutations, R1448H and R1448C, substitute histidine and cysteine for arginine in the S4 segment of domain 4. These mutations, expressed in a cell line, have only small effects on the activation of Na+ currents, but mutant channels inactivate more slowly with less voltage dependence than wild-type channels and exhibit an enhanced rate of recovery from inactivation. Increase of extracellular pH made the rate of inactivation of R1448H similar to that of R1448C, suggesting that this residue has an extracellular location and that its charge is important for normal inactivation. Analysis of single-channel data reveals that mutant channels inactivate normally from closed states, but poorly from the open state. The data suggest a critical role for the S4 helix of domain 4 in coupling between activation and inactivation.

Honeybee locomotion is impaired by Am-CaV3 low voltage-activated Ca2+ channel antagonist.

Voltage-gated Ca2+ channels are key transducers of cellular excitability and participate in several crucial physiological responses. In vertebrates, 10 Ca2+ channel genes, grouped in 3 families (CaV1, CaV2 and CaV3), have been described and characterized. Insects possess only one member of each family. These genes have been isolated in a limited number of species and very few have been characterized although, in addition to their crucial role, they may represent a collateral target for neurotoxic insecticides. We have isolated the 3 genes coding for the 3 Ca2+ channels expressed in Apis mellifera. This work provides the first detailed characterization of the honeybee T-type CaV3 Ca2+ channel and demonstrates the low toxicity of inhibiting this channel. Comparing Ca2+ currents recorded in bee neurons and myocytes with Ca2+ currents recorded in Xenopus oocytes expressing the honeybee CaV3 gene suggests native expression in bee muscle cells only. High-voltage activated Ca2+ channels could be recorded in the somata of different cultured bee neurons. These functional data were confirmed by in situ hybridization, immunolocalization and in vivo analysis of the effects of a CaV3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of CaV3 suggest a role in honeybee muscle function.

Novel mechanism for Brugada syndrome: defective surface localization of an SCN5A mutant (R1432G).

The SCN5A gene encodes the alpha subunit of the human heart sodium channel (hH1), which plays a critical role in cardiac excitability. Mutations of SCN5A underlie Brugada syndrome, an inherited disorder that leads to ventricular fibrillation and sudden death. This study describes changes in cellular localization and functional expression of hH1 in a naturally occurring SCN5A mutation (R1432G) reported for Brugada syndrome. Using patch-clamp experiments, we show that there is an abolition of functional hH1 expression in R1432G mutants expressed in human tsA201 cells but not in Xenopus oocytes. In tsA201 cells, a conservative positively charged mutant, R1432K, produced sodium currents with normal gating properties, whereas other mutations at this site abolished functional sodium channel expression. Immunofluorescent staining and confocal microscopy showed that the wild-type alpha subunit expressed in tsA201 cells was localized to the cell surface, whereas the R1432G mutant was colocalized with calnexin within the endoplasmic reticulum. The beta(1) subunit was also localized to the cell surface in the presence of the alpha subunit; however, in its absence, the beta(1) subunit was restricted to a perinuclear localization. These results demonstrate that the disruption of SCN5A cell-surface localization is one mechanism that can account for the loss of functional sodium channels in Brugada syndrome. The full text of this article is available at http://www.circresaha.org.

Homozygous SCN5A mutation in long-QT syndrome with functional two-to-one atrioventricular block.

Heterozygous mutations in genes encoding cardiac ionic channel subunits KCNQ1, HERG, SCN5A, KCNE1, and KCNE2 are causally involved in the dominant form of long-QT syndrome (LQTS) while homozygous mutations in KCNQ1 and KCNE1 cause LQTS with or without congenital deafness. In addition, two homozygous HERG mutations have been associated with severe LQTS with functional atrioventricular conduction anomalies in young children. A 2:1 atrioventricular block (AVB) with a major QTc prolongation (526 ms) was evidenced in a 5-year-old boy referred for syncope and seizure. LQTS was diagnosed and beta-blocking therapy initiated leading to normal atrioventricular conduction. Electrophysiological study provided support that location of the AVB was infra-Hisian. DNA analysis was performed in the proband and in asymptomatic family members. A novel missense mutation, V1777M, in the early C-terminal domain of SCN5A was identified. The proband was homozygous while the parents and two siblings were heterozygous carriers. Homozygote and heterozygote expression of the mutant channels in tsA201 mammalian cells resulted in a persistent inward sodium current of 3.96+/-0.83% and 1.49+/-0.47% at -30 mV, respectively, which was dramatically reduced in the presence of tetrodotoxin. This study provides the first evidence for a homozygous missense mutation in SCN5A and suggests that LQTS with functional 2:1 AVB in young children, a severe phenotype associated with bad prognosis, may be caused by homozygous or heterozygous compound mutations not only in HERG but also in SCN5A. The full text of this article is available at http://www.circresaha.org.

Gating properties of Na(v)1.7 and Na(v)1.8 peripheral nerve sodium channels.

Several distinct components of voltage-gated sodium current have been recorded from native dorsal root ganglion (DRG) neurons that display differences in gating and pharmacology. This study compares the electrophysiological properties of two peripheral nerve sodium channels that are expressed selectively in DRG neurons (Na(v)1.7 and Na(v)1.8). Recombinant Na(v)1.7 and Na(v)1.8 sodium channels were coexpressed with the auxiliary beta(1) subunit in Xenopus oocytes. In this system coexpression of the beta(1) subunit with Na(v)1.7 and Na(v)1.8 channels results in more rapid inactivation, a shift in midpoints of steady-state activation and inactivation to more hyperpolarizing potentials, and an acceleration of recovery from inactivation. The coinjection of beta(1) subunit also significantly increases the expression of Na(v)1.8 by sixfold but has no effect on the expression of Na(v)1.7. In addition, a great percentage of Na(v)1.8+beta(1) channels is observed to enter rapidly into the slow inactivated states, in contrast to Nav1.7+beta(1) channels. Consequently, the rapid entry into slow inactivation is believed to cause a frequency-dependent reduction of Na(v)1.8+beta(1) channel amplitudes, seen during repetitive pulsing between 1 and 2 Hz. However, at higher frequencies (>20 Hz) Na(v)1.8+beta(1) channels reach a steady state to approximately 42% of total current. The presence of this steady-state sodium channel activity, coupled with the high activation threshold (V(0.5) = -3.3 mV) of Na(v)1.8+beta(1), could enable the nociceptive fibers to fire spontaneously after nerve injury.

Cysteine scanning analysis of the IFM cluster in the inactivation gate of a human heart sodium channel.

UNLABELLED: The conserved isoleucine-phenylalanine-methionine (IFM) hydrophobic cluster located in the III-IV linker of voltage-gated sodium channels has been identified as a major component of the fast inactivation gate in these channels. OBJECTIVES: The aim of our study was to probe the contribution of each amino acids of the IFM cluster to the inactivation. METHODS: A combination of site-directed mutagenesis, cysteine covalent modification and electrophysiological recording techniques were used to elucidate the role of isoleucine1485 and methionine1487 on hH1 sodium channels expressed in tsA201 cells. RESULTS: Mutant I1485C behaves like mutant F1486C studied earlier: producing an incomplete inactivation (residual current), a slowing and change in the voltage-dependence of the time constants of current decay, a shift of the steady-state inactivation to more depolarized voltages, and a faster recovery from inactivation than the wild-type hH1. The electrophysiological parameters of mutant M1487C are similar to those of wild-type hH1 except for the presence of a residual current. Exposure of the cytoplasmic surface of the mutants to MTS reagents MTSES, MTSET and MTSBn further disrupted inactivation. In order to explain differences in the amplitude of the sustained currents recorded in the presence of MTSES or MTSET, we studied the effects of exposure of mutants 11485C, F1486C and M1487C to acidic and basic pH in the absence and presence of MTSES and MTSET. The effects of MTSES [negatively charged (-)] and MTSET (+) on the amplitude of the residual current of mutant F1486C were modulated by changes in intracellular pH. CONCLUSION: Isoleucine1487 and methionine1485, which surround phenylalanine1487 contribute to stabilizing the inactivation particle for fast inactivation.

Electrophysiological characterization of SCN5A mutations causing long QT (E1784K) and Brugada (R1512W and R1432G) syndromes.

UNLABELLED: Familial long QT syndrome (LQTS) and Brugada syndrome are two distinct human hereditary cardiac diseases known to cause ventricular tachyarrhythmias (torsade de pointes) and idiopathic ventricular fibrillation, respectively, which can both lead to sudden death. OBJECTIVE: In this study we have identified and electrophysiologically characterized, in patients having either LQTS or Brugada syndrome, three mutations in SCN5A (a cardiac sodium channel gene). METHOD: The mutant channels were expressed in a mammalian expression system and studied by means of the patch clamp technique. RESULTS: The R1512W mutation found in our first patient diagnosed with Brugada syndrome produced a slowing of both inactivation and recovery from inactivation. The R4132G mutation found in our second patient who also presented Brugada syndrome, resulted in no measurable sodium currents. Both Brugada syndrome patients showed ST segment elevation and right bundle-branch block, and had experienced syncopes. The E1784K mutation found in the LQTS showed a persistent inward sodium current, a hyperpolarized shift of the steady-sate inactivation and a faster recovery from inactivation. CONCLUSION: The different clinical manifestations of these three mutations most probably originate from the distinct electrophysiological abnormalities of the mutant cardiac sodium channels reported in this study.

Ca2+ oscillations induced by histamine H1 receptor stimulation in HeLa cells: Fura-2 and patch clamp analysis.

The response of HeLa cells to histamine H1 receptor stimulation is characterized by periodic increases in cytosolic free Ca2+ concentration. The mechanisms underlying this oscillatory behaviour are not well understood. Fura-2 and patch clamp experiments carried out on HeLa cells have previously shown: (a) that Ca2+ oscillations are not initially dependent on the presence of external Ca2+, that external Ca2+ is required to maintain the oscillatory activity; (b) that a depolarization of the cell membrane leads to an inhibition of Ca2+ oscillations during the external Ca2+ dependent phase of the process; and (c) that Ca2+ oscillations can be abolished during this latter phase by the exogenous addition of Ca2+ channel blocking agents, such as Co2+ or La3+. The contribution of the inositol phosphate pathway to Ca2+ oscillations was more recently investigated in whole cell experiments performed with patch pipettes containing IP3 or the non-hydrolysable GTP analogue GTP-gamma S. Clear periodic current fluctuations were recorded using both patch pipette solutions. Assuming that the intracellular IP3 level remained constant under these conditions, these findings provide direct evidence that the Ca2+ oscillations in HeLa cells do not arise from a periodic production of IP3. The effect of the internal and external cell pH on the oscillatory process was also investigated in Fura-2 and patch clamp experiments. It was found that an increase in intracellular pH from 7.4 to 7.7 during the external Ca2+ dependent phase of the histamine stimulation abolishes the appearance of Ca2+ spikes whereas, a cellular acidification to pH 7.2 maintains or stimulates the Ca2+ oscillatory activity. The former effect was observed in the absence of Ca2+ in the bathing medium, indicating that the inhibitory action of alkaline pH was not related to a reduced Ca2+ entry. An increase in extracellular pH from 7.3 to 9.0 in contrast elicited an intracellular Ca2+ accumulation which resulted in most cases in an inhibition of the oscillatory process. This effect was dependent on external Ca2+ and was observed in alkaline internal pH conditions (pH 7.7). These observations suggest: (a) that the net Ca2+ influx in HeLa cells is strongly dependent on the cell internal and external pH; and (b) that the magnitude of this Ca2+ influx controls to a large extent the oscillation frequency. Finally, an inhibition of the histamine induced Ca2+ oscillatory activity was observed following the addition of the Ca(2+)-induced Ca(2+)-release (CICR) inhibitor adenine to the external medium.(ABSTRACT TRUNCATED AT 250 WORDS)