Large Conductance Voltage- and Ca2+-gated Potassium (BK) Channel β4 Subunit Influences Sensitivity and Tolerance to Alcohol by Altering Its Response to Kinases

Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca²⁺-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the β4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αβ4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the β4 subunit on tolerance may result from a striking effect of β4 on kinase modulation of the BK channel. We examine the influence of the β4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the β4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the β4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol.     

Chaotic mixer improves microarray hybridization

Hybridization is an important aspect of microarray experimental design which influences array signal levels and the repeatability of data within an array and across different arrays. Current methods typically require 24h and use target inefficiently. In these studies, we compare hybridization signals obtained in conventional static hybridization, which depends on diffusional target delivery, with signals obtained in a dynamic hybridization chamber, which employs a fluid mixer based on chaotic advection theory to deliver targets across a conventional glass slide array. Microarrays were printed with a pattern of 102 identical probe spots containing a 65-mer oligonucleotide capture probe. Hybridization of a 725-bp fluorescently labeled target was used to measure average target hybridization levels, local signal-to-noise ratios, and array hybridization uniformity. Dynamic hybridization for 1h with 1 or 10ng of target DNA increased hybridization signal intensities approximately threefold over a 24-h static hybridization. Similarly, a 10- or 60-min dynamic hybridization of 10ng of target DNA increased hybridization signal intensities fourfold over a 24h static hybridization. In time course studies, static hybridization reached a maximum within 8 to 12h using either 1 or 10ng of target. In time course studies using the dynamic hybridization chamber, hybridization using 1ng of target increased to a maximum at 4h and that using 10ng of target did not vary over the time points tested. In comparison to static hybridization, dynamic hybridization reduced the signal-to-noise ratios threefold and reduced spot-to-spot variation twofold. Therefore, we conclude that dynamic hybridization based on a chaotic mixer design improves both the speed of hybridization and the maximum level of hybridization while increasing signal-to-noise ratios and reducing spot-to-spot variation.     

Immunohistochemical staining of endomorphin 1 and 2 in the immune cells of the spleen

Endomorphin 1 (EM-1) and EM-2 have been widely reported in the cells of the central nervous system (CNS) but limited research has been done regarding their distribution in the peripheral system. The occurrence of EM-1 and -2 in the spleen as measured by RIA and their ability to mediate immune function imply a role for EMs in this area. The current study examines the localization of EM-1 and -2 in the immune cells of the spleen of male and female rats via an immunohistochemical procedure. In both genders, EM-1 and -2 immunoreactive staining was predominantly present in macrophages and B cells with minimal EM immunoreactive staining in T cells. This is the first evidence of a differential distribution of EM-1 and -2 in cells of the immune system.     

Non-secretory solitary plasmacytoma of the spleen

A case of primary nonsecretory plasmacytoma of the spleen is reported. On laparotomy and splenectomy a 920 g spleen was removed, measuring 16×14×6 cm. The cut surface of the entire spleen showed that the tumour occupied most of the splenic tissue. A bone marrow aspirate and trephine, skeletal survey showed no signs of myeloma. Biopsy of the liver and regional lymph nodes was normal. Immunocytochemistry of the splenic tumour showed positivity for pan-B and plasma cell markers. After splenectomy the patient was treated with chemotherapy according to protocol VBCMP (M2).     

Incidence and Risk Factors for Neonatal Tetanus in Admissions to Kilifi County Hospital,Kenya

Background

Neonatal Tetanus (NT) is a preventable cause of mortality and neurological sequelae that occurs at higher incidence in resource-poor countries, presumably because of low maternal immunisation rates and unhygienic cord care practices. We aimed to determine changes in the incidence of NT, characterize and investigate the associated risk factors and mortality in a prospective cohort study including all admissions over a 15-year period at a County hospital on the Kenyan coast, a region with relatively high historical NT rates within Kenya.

Methods

We assessed all neonatal admissions to Kilifi County Hospital in Kenya (1999–2013) and identified cases of NT (standard clinical case definition) admitted during this time. Poisson regression was used to examine change in incidence of NT using accurate denominator data from an area of active demographic surveillance. Logistic regression was used to investigate the risk factors for NT and factors associated with mortality in NT amongst neonatal admissions. A subset of sera from mothers (n = 61) and neonates (n = 47) were tested for anti-tetanus antibodies.

Results

There were 191 NT admissions, of whom 187 (98%) were home deliveries. Incidence of NT declined significantly (Incidence Rate Ratio: 0.85 (95% Confidence interval 0.81–0.89), P<0.001) but the case fatality (62%) did not change over the study period (P = 0.536). Younger infant age at admission (P = 0.001) was the only independent predictor of mortality. Compared to neonatal hospital admittee controls, the proportion of home births was higher among the cases. Sera tested for antitetanus antibodies showed most mothers (50/61, 82%) had undetectable levels of antitetanus antibodies, and most (8/9, 89%) mothers with detectable antibodies had a neonate without protective levels.

Conclusions

Incidence of NT in Kilifi County has significantly reduced, with reductions following immunisation campaigns. Our results suggest immunisation efforts are effective if sustained and efforts should continue to expand coverage.     

Catalase as a source of both X- and V-factor for Haemophilus influenzae

Haemophilus influenzae requires two growth factors, designated factor X (porphyrin) and factor V (NAD). Mammalian catalases contain both bound heme and NADPH. This study shows that catalase can supply both factors X and V to H. influenzae in vitro, thus representing a potential in vivo source of these essential growth factors.