Linkage investigation of a large family with Reifenstein''s syndrome

Linkage analysis of a 116-member family with 11 males affected by Reifenstein's syndrome is reported. One X-chromosomal and eight autosomal markers were used. Close linkage can be excluded for P, K, MNS and Xg-a. A possibility of close linkage to ABO is discussed.     

Rga (Rodgers) and the HLA Region: Linkage and Associations

In 19 families with 97 children the segregation of Rg^a (Rodgers) was found to be compatible with Mendelian inheritance and five backcross and 14 intercross families were found among HLA and Bf typed families. Close linkage (lods + 17.82) without recombination was found between Rg and the HLA region, with a direct count of 96 nonrecombinant meioses for Rg-HLA-B. Kg- was strongly associated with HLA-B8 (29 of 30 haplotypes) and probably associated with Bw40, but did occur on other HLA-B haplotypes. By inference .Rg^- is negatively associated with Ch^- (Chido). The Rg^-Ch^- haplotype has not been observed. Rg³ and Ch^a may or may not be coded for by different sites of the same cistron closely linked to HLA-B:C and cannot as yet be excluded from being parts of B or C.     

Rga (Rodgers) and the HLA Region: Linkage and Associations

In 19 families with 97 children the segregation of Rg^a (Rodgers) was found to be compatible with Mendelian inheritance and five backcross and 14 intercross families were found among HLA and Bf typed families. Close linkage (lods + 17.82) without recombination was found between Rg and the HLA region, with a direct count of 96 nonrecombinant meioses for Rg-HLA-B. Kg- was strongly associated with HLA-B8 (29 of 30 haplotypes) and probably associated with Bw40, but did occur on other HLA-B haplotypes. By inference.Rg^- is negatively associated with Ch^- (Chido). The Rg^-Ch^- haplotype has not been observed. Rg³ and Ch^a may or may not be coded for by different sites of the same cistron closely linked to HLA-B:C and cannot as yet be excluded from being parts of B or C.     

Idiopathic and secondary membranous nephropathy and polymorphism at TAP1 and HLA-DMA loci

In a previous study on the effects of TAP1 and TAP2 gene polymorphism in kidney allograft recipients, we found no association between graft outcome and recipient/donor TAP1 and TAP2 allele polymorphism or compatibility, but we observed a surprising increased frequency of the TAP1*0201 allele among kidney recipients. This increase was restricted to patients with glomerulopathy. We now report on a larger cohort of 178 patients with membranous nephropathy who were typed for their HLA-DPB1, -DRB1, -DMA, -DMB, LMP2, LMP, TAP1 and TAP2 genes compared with 100 random ethnically matched and healthy unrelated individuals used as controls. The results show a significant increased frequency of two markers in membranous nephropathy patients as compared with controls: firstly the previously recognized increase in HLA-DR3 (59% vs 18%: P_c < 1 × 10^-9, RR=6.6), secondly a new association with two TAP1 amino acid variants displaying respectively a valine in amino acid position 333 (TAP1-Val^-333) and consequently a glycine in position 637 (TAP1Gly^-637) due to its strong linkage disequilibrium with Val^-333. No linkage disequilibrium was found between TAP1-Val^-333 and HLA-DR3. Moreover, we also noticed a decrease of the DMA*0102 phenotype in membranous nephropathy patients. The other HLA-DPB, -DMB, LMP2, LMP7 and TAP2 phenotype frequencies were roughly similar between patients and controls. These results show that the TAP1-Val^-333 like HLA-DR3 phenotype is positively associated with membranous nephropathy and that these two risk factors are not cumulative in membranous nephropathy pathophysiology.     

Allelic association discriminates draft orders

A year ago there was hope that a finished sequence of the human genome would soon be publicly available and would give a more reliable locus order than an unconstrained radiation hybrid or genetic map. Alas, there are now different draft orders for each region, none of which may be correct because of gaps, uncertain polarity of contigs, and errors in assembly. Shortly before these drafts became available, we analysed allelic association (also called linkage disequilibrium, LD) in the FRAX region in a large sample of haplotypes (Ennis et al . 2000). We demonstrate here that this material discriminates among alternative draft orders. To express support for discrimination between two values of χ²=−2 ln L we use the Akaike criterion AIC = df[χ²/min χ²−1]. Excluding premutations and full mutations at FMR1, all maps have 715 degrees of freedom (df) among 717 pairs of alleles after accepting L = 0 and estimating M, ∈ in the Malecot equation E(ρ) = Me ^−∈d, where ρ is the association between a pair of alleles at distance d. An AIC in excess of 2 provides evidence against a map with the larger χ².     

Phenotypical Characterization of Cells in the Thoracic Duct of Patients with and without Systemic Inflammatory Response Syndrome and Multiple Organ Failure

The subset composition and recirculation properties of the migrating lymphocyte pool in humans is largely unknown. The present study was conducted in order to phenotypically characterize cells in human thoracic duct lymph of patients under non-inflammatory and inflammatory conditions. These data were compared with data from peripheral blood, with special emphasis on those cells homing to the gut. Thoracic duct lymph and peripheral blood contained comparable proportions of B and T lymphocytes and CD8⁺ cells. Thoracic duct lymph contained proportionally more CD4⁺ cells, more CD4⁺CD45RO⁺ that express α₄β₇ cells and more CD8⁺CD45RO⁺ that express α₄β₇, as compared to peripheral blood. These data suggest an equal recirculation rate of B and T lymphocytes; a more active recirculation of CD4⁺ cells compared to CD8⁺ cells; and a more active recirculation of memory cells to the gut as compared to other extra-lymphoid sites in patients under non-inflammatory conditions. Data were also obtained in patients with the system inflammatory response syndrome and multiple organ failure. Although it is generally assumed that granulocytes and monocytes do not recirculate, lymph of multiple organ failure patients contained significantly more granulocytes than monocytes, indicating that in severe generalized inflammatory states these cells re-enter the circulation through the thoracic duct. Furthermore, no increased activation of cells homing to the gut was found in these patients.     

Family studies on nucleoside phosphorylase and the short arm of chromosome 14

A family with two nucleoside phosphorylase-deficient patients has been scored for the segregation of NP⁰ and the variable region 14p. The most likely 14p: NP recombination fraction is (M5 in males and 0–30 in females.
There is no family data to assign the Pi:Gm linkage group to chromosome 14, but as immunoglobulin heavy chain has been assigned to this chromosome by somatic cell methods the most likely gene order is 14p:NP: Pi:Gm with Pi in 14q2 and Gm in 14(q23 →q32), but the order 14p:NP:Gm:Pi with Pi in 14(q24 → qter) and Gm in 14(q22 → q24) is not excluded.
The available linkage data between biochemical markers on acrocentric chromosomes and their short arm markers suggest that there may be more recombination towards the ends of human chromosomes whether or not those ends carry centromeres.     

Temperature enhances exocytosis efficiency at the mouse inner hair cell ribbon synapse

Hearing relies on fast and sustained neurotransmitter release from inner hair cells (IHCs) onto the afferent auditory nerve fibres. The temperature dependence of Ca²⁺ current and transmitter release at the IHCs ribbon synapse has not been investigated thus far. To assess the influence of temperature on calcium-triggered exocytosis, patch-clamp recordings of voltage-gated L-type Ca²⁺ influx and exocytic membrane capacitance changes were performed at room (25°C) and physiological (35–37°C) temperatures. An increase in temperature within this range increased the L-type Ca²⁺ current amplitude of IHCs ( Q ₁₀= 1.3) and accelerates the activation kinetics. Fast exocytosis, probed by 20 ms depolarization, was enhanced at physiological temperature with a Q ₁₀ of 2.1. The amplitude of fast release was elevated disproportionately to the increase in Ca²⁺ influx. In contrast, the rate of sustained exocytosis (exocytic rate between 20 and 100 ms of depolarization) did not show a significant increase at physiological temperature. Altogether, these data indicate that the efficiency of fast exocytosis is higher at physiological temperature than at room temperature and suggest that the number of readily releasable vesicles available at the active zone is higher at physiological temperature.     

Defective interaction between dual oscillators for respiratory rhythm generation in Na+,K+-ATPase α2 subunit-deficient mice

The current concept regarding the respiratory centre in mammals is that it is composed of two distinct rhythm-generating neuronal networks in the ventrolateral medulla. These two rhythm generators can be active independently but are normally coupled in newborn and juvenile rats. Detailed characteristics of each generator and the neuronal mechanisms of coupling during development remain to be elucidated. Here, we report a knockout mouse (Na⁺,K⁺-ATPase α2 subunit gene ( Atp1a2 ) knockout) that may be defective in functional coupling between the two respiration-related rhythm generators. We investigated respiration-related neuron activity in an en bloc brainstem–spinal cord preparation isolated from embryonic day 18.5 Atp1a2^{−/ −} mouse fetuses. In the presence of adrenaline, two different types of rhythm generators were identified. One produced inspiratory burst activity that correlated with C4 inspiratory activity and was thought to be the inspiratory rhythm generator on the basis of its location and sensitivity to a μ-opiate receptor agonist, [ d -Ala2, N -Me-Phe4, Gly5-ol]-enkephalin (DAMGO). The other was presumed to be the preinspiratory rhythm generator because it was insensitive to DAMGO and correlated with facial nerve activity. Coupling between these rhythm generators did not function in the normal manner in Atp1a2^{−/ −} mice, as shown by disruption of the linkage between the preinspiratory burst and the inspiratory burst. Coupling was partially restored by repeated activation of the neurons within the networks, suggesting the involvement of an activity-dependent process in the prenatal development of this coupling.     

Calmodulin binding to M-type K+ channels assayed by TIRF/FRET in living cells

Calmodulin (CaM) binds to KCNQ2–4 channels within their carboxy termini, where it regulates channel function. The existing data have not resolved the Ca²⁺ dependence of the interaction between the channels and CaM. We performed glutathione S-transferase (GST)-pull-down assays between purified KCNQ2–4 carboxy termini and CaM proteins to determine the Ca²⁺ dependence of the interaction in vitro . The assays showed substantial Ca²⁺ dependence of the interaction of the channels with wild-type (WT) CaM, but not with dominant-negative (DN) CaM. To demonstrate CaM–channel interactions in individual living cells, we performed fluorescence resonance energy transfer (FRET) between ECFP-tagged KCNQ2–4 channels and EYFP-tagged CaM expressed in CHO cells, performed under total internal reflection fluorescence (TIRF) microscopy, in which excitation light only penetrates several hundred nanometres into the cell, thus isolating membrane events. FRET was assayed between the channels and either WT or DN CaM, performed under conditions of normal [Ca²⁺]_i, low [Ca²⁺]_i or high [Ca²⁺]_i induced by empirically optimized bathing solutions. The FRET data suggest a strong Ca²⁺ dependence for the interaction between WT CaM and KCNQ2, but less so for KCNQ3 and KCNQ4. FRET between all KCNQ2–4 channels and DN CaM was robust, and not significantly Ca²⁺ dependent. These data show interactions between CaM and KCNQ channels in living cells, and suggest that the interactions between KCNQ2–4 channels and CaM are likely to have Ca²⁺-dependent and Ca²⁺-independent components.     

Distribution of HLA-DQA1 and -DQB1 alleles and DQA1-DQB1 genotypes among Senegalese patients with insulin-dependent diabetes mellitus

Transracial analysis is one method for distinguishing primary associations between insulin-dependent diabetes mellitus (IDDM) and HLA II alleles from those related to linkage disequilibrium. Black people have different DR-DQ relationships from other races and are a useful group to investigate HLA-D regions associated with IDDM. In this study, we compared the frequencies of HLA-DQA1 and DQB1 alleles in Senegalese IDDM and control subjects. DQA1*0301 was positively associated with insulin-dependent diabetes mellitus (p<10^-9, OR 5.21), as were DQB1*0201 and *0302 (p<10^-7 OR=3.55, p<10^-3 OR=3.20, respectively). The positive associations with DQA1*0301, DQB1*0201 and DQB1*0302 are consistent with all racial groups investigated. However, taken together, the data in Senegalese population show that susceptibility and resistance to IDDM are associated both with particular haplotypes and DQA1-DQB1 heterodimers.     

Mini-dystrophin restores L-type calcium currents in skeletal muscle of transgenic mdx mice

L-type calcium currents ( i _Ca) were recorded using the two-microelectrode voltage-clamp technique in single short toe muscle fibres of three different mouse strains: (i) C57/SV129 wild-type mice (wt); (ii) mdx mice (an animal model for Duchenne muscular dystrophy; and (iii) transgenically engineered mini-dystrophin (MinD)-expressing mdx mice. The activation and inactivation properties of i _Ca were examined in 2- to 18-month-old animals. Ca²⁺ current densities at 0 mV in mdx fibres increased with age, but were always significantly smaller compared to age-matched wild-type fibres. Time-to-peak (TTP) of i _Ca was prolonged in mdx fibres compared to wt fibres. MinD fibres always showed similar TTP and current amplitudes compared to age-matched wt fibres. In all three genotypes, the voltage-dependent inactivation and deactivation of i _Ca were similar. Intracellular resting calcium concentration ([Ca²⁺]_i) and the distribution of dihydropyridine binding sites were also not different in young animals of all three genotypes, whereas i _Ca was markedly reduced in mdx fibres. We conclude, that dystrophin influences L-type Ca²⁺ channels via a direct or indirect linkage which may be disrupted in mdx mice and may be crucial for proper excitation–contraction coupling initiating Ca²⁺ release from the sarcoplasmic reticulum. This linkage seems to be fully restored in the presence of mini-dystrophin.     

Terminal deletion(4)(q33) in a male infant

The deletion of 4q31→qter is associated with a recognizable "4q^- syndrome". It has been proposed that the much rarer deletion 4q33→qter causes a milder phenotypic expression of the 4q^- syndrome. We present the second case, the first male, with the latter deletion and compare his clinical features to those of other 4q^- patients.     

Physiological roles of ATP-sensitive K+ channels in smooth muscle

Potassium channels that are inhibited by intracellular ATP (ATP_i) were first identified in ventricular myocytes, and are referred to as ATP-sensitive K⁺ channels (i.e. K_ATP channels). Subsequently, K⁺ channels with similar characteristics have been demonstrated in many other tissues (pancreatic β-cells, skeletal muscle, central neurones, smooth muscle). Approximately one decade ago, K_ATP channels were cloned and were found to be composed of at least two subunits: an inwardly rectifying K⁺ channel six family (Kir6.x) that forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR) that accounts for several pharmacological properties. Various types of native K_ATP channels have been identified in a number of visceral and vascular smooth muscles in single-channel recordings. However, little attention has been paid to the molecular properties of the subunits in K_ATP channels and it is important to determine the relative expression of K_ATP channel components which give rise to native K_ATP channels in smooth muscle. The aim of this review is to briefly discuss the current knowledge available for K_ATP channels with the main interest in the molecular basis of native K_ATP channels, and to discuss their possible linkage with physiological functions in smooth muscle.     

Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca²⁺-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between −65 and −40 mV and spontaneous waves evoked by increasing external Ca²⁺ concentration to 4 m m . Application of 100 μ m TBQ decreased the frequency of waves. This was associated with increases of resting [Ca²⁺]_i, the time constant of decay of [Ca²⁺]_i and the integral of the accompanying Na⁺–Ca²⁺ exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca²⁺ efflux per wave. The SR Ca²⁺ content when a wave was about to propagate decreased to 91.7 ± 3.2%. The period between waves increased in direct proportion to the Ca²⁺ efflux per wave meaning that TBQ had no effect on the Ca²⁺ efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca²⁺ pumping by SERCA between waves but, rather, to more Ca²⁺ loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca²⁺ release propagating at a given SR content.     

The role of the sarcoplasmic reticulum in neonatal uterine smooth muscle: enhanced role compared to adult rat

Little is known about contractile activity, response to agonists or excitation-contraction coupling in neonatal smooth muscle. We have therefore investigated 10-day rat uterus to better understand these processes, and compared it to adult uterus to elucidate how control of contractility develops. Spontaneous contractions are present in the 10-day neonatal uterus, although they are not as large or as regular as those present in adult tissues. External Ca²⁺ entry via L-type Ca²⁺ channels is the sole source of Ca²⁺ and is essential for the spontaneous activity. The neonatal uterus was responsive to carbachol or prostaglandin F_2α application; it showed a marked stimulation and a clear dissociation between the force and Ca²⁺ changes. Such sensitization was not apparent in adult rat myometrium. The sarcoplasmic reticulum (SR) had more releasable Ca²⁺ and contributed more to the response to agonists in neonatal compared to adult tissues. Thus, Ca²⁺ entry as opposed to SR Ca²⁺ release contributed much less to the uterine response to agonists in the neonatal, compared to adult tissues. Inhibition of the SR by cyclopiazonic acid also caused a more vigorous increase in Ca²⁺ and contractile activity, particularly frequency, in the neonatal compared to the adult uterus. Taken together these data suggest that: (1) spontaneous activity is already present by day 10, (2) receptor-coupling and excitation-contraction signalling pathways are functional, (3) the SR and Ca²⁺ sensitization mechanisms play a more prominent role in the neonate, and (4) there is a shift to a greater reliance on Ca²⁺ entry and excitability with development of the myometrium.     

Structurally delineating stromal interaction molecules as the endoplasmic reticulum calcium sensors and regulators of calcium release-activated calcium entry

Summary:  The endoplasmic reticulum (ER) lumen stores a crucial source of calcium (Ca²⁺) maintained orders of magnitude higher than the cytosol for the activation of a plethora of cellular responses transmitted in health and disease by a mutually efficient and communicative exchange of Ca²⁺ between compartments. A coordination of the Ca²⁺ signal is evident in the development of Ca²⁺ release-activated Ca²⁺ (CRAC) entry, vital to lymphocyte activation and replenishing of the ER Ca²⁺ stores, where modest decreases in ER luminal Ca²⁺ induce sustained increases in cytosolic Ca²⁺ sourced from steadfast extracellular Ca²⁺ supplies. While protein sensors that transduce Ca²⁺ signals in the cytosol such as calmodulin are succinctly understood, comparative data on the ER luminal Ca²⁺ sensors is only recently coming to light with the discovery that stromal interaction molecules (STIMs) sense variations in ER stored Ca²⁺ levels in the functional regulation of plasma membrane Orai proteins, the major component of CRAC channel pores. Drawing from data on the role of STIMs in the modulation of CRAC entry, this review illustrates the structural features that delimit the functional characteristics of ER Ca²⁺ sensors relative to well known cytoplasmic Ca²⁺ sensors.     

Mechanisms underlying variations in excitation–contraction coupling across the mouse left ventricular free wall

Ca²⁺ release during excitation–contraction (EC) coupling varies across the left ventricular free wall. Here, we investigated the mechanisms underlying EC coupling differences between mouse left ventricular epicardial (Epi) and endocardial (Endo) myocytes. We found that diastolic and systolic [Ca²⁺]_i was higher in paced Endo than in Epi myocytes. Our data indicated that differences in action potential (AP) waveform between Epi and Endo cells only partially accounted for differences in [Ca²⁺]_i. Rather, we found that the amplitude of the [Ca²⁺]_i transient, but not its trigger – the Ca²⁺ current – was larger in Endo than in Epi cells. We also found that spontaneous Ca²⁺ spark activity was about 2.8-fold higher in Endo than in Epi cells. Interestingly, ryanodine receptor type 2 (RyR2) protein expression was nearly 2-fold higher in Endo than in Epi myocytes. Finally, we observed less Na⁺–Ca²⁺ exchanger function in Endo than in Epi cells, which was associated with decreased Ca²⁺ efflux during the AP; this contributed to higher diastolic [Ca²⁺]_i and SR Ca²⁺ in Endo than in Epi cells during pacing. We propose that transmural differences in AP waveform, SR Ca²⁺ release, and Na⁺–Ca²⁺ exchanger function underlie differences in [Ca²⁺]_i and EC coupling across the left ventricular free wall.     

Association of angiotensin II type 1 receptor gene polymorphism with essential hypertension

The renin-angiotensin system is involved in control of blood pressure and salt and fluid homeostasis. Genes for components of this system have been of major focus in research on the causation of the common, complex, polygenic trait, essential hypertension (HT). Association of an A→C variant at nucleotide 1166 of the angiotensin II type 1 receptor (AT₁R) gene with HT, but an absence of linkage of this locus with this disease, has been reported recently. Since confirmation in a different setting is imperative, we performed a cross-sectional case-control study of the A1166C variant in a well-characterized group of 108 Caucasian HT subjects with a strong family history (two affected parents) and early onset disease. Genotyping was by mismatch polymerase chain reaction/ Bfr I restriction fragment length polymorphism analysis. Frequency of the C¹¹⁶⁶allele was 0.40 in HTs and 0.29 in normotensives. The difference in genotype (χ²= 13, P = 0.0015) and allele (χ²= 5.3, P = 0.02) frequencies between the two groups was significant (odds ratio for CC vs AA+AC = 7.3 [95% CI, 1.9–31.9). The present results implicate the AT₁R gene, or a locus in linkage disequilibrium with the variant tested, in the causation of essential HT.     

Immunization with the Light Chain and the VL Domain of the Isologous Myeloma Protein 315 Inhibits Growth of Mouse Plasmacytoma MOPC315

Prior immunization of BALB/c mice with free light chains from myeloma proein 315 (V_L³¹⁵) and its variable domain (V_L^3l5) inhibited the growth of subcutaneously injected MOPC315 tumour cells. The growth suppression observed after immunization with L³¹⁵ was equivalant to that which resulted from immunization with the complete M315. V_L³¹⁵ and non-polymerized L³¹⁵ did not elicit specific antibodies. Polymerized L³¹⁵; induced both suppression of MOPC315 growth and antibodies specific for free L³¹⁵; however, these anybodies did not rect with the complete M315, nor were they absorbed by MOPC315 tumour cells. the data indicated that the suppression of tumour growth was mediated by specifically sensitized cells acting in the absence of antibodies against M3l5 or L³¹⁵. Immunization with the variable domain of the heavy chain from M315 (V_H³¹⁵) had no effect on the growth of MOPC315, the M315 fragment and subunits that induced growth suppression were thus identical with those capable of inducing T helper cells in BALB/c mice.