Mutation analysis of feline Niemann-Pick C1 disease

Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.     

The advantage of mucosal immunization for polysaccharide-specific memory responses in early life

The aim of vaccination is to rapidly elicit protective immunity and generate memory for sustained protection. We studied the induction and persistence of polysaccharide (PS)-specific memory in neonatal and infant mice primed with pneumococcal conjugate (Pnc1-TT) by assessing the response to native pneumococcal PS (PPS-1), the kinetics of the PPS-1-specific IgG response to a second Pnc1-TT dose and affinity maturation. A subcutaneous (s.c.) Pnc1-TT booster induced a rapid increase in PPS-1-specific IgG, indicating efficient priming for memory by a single dose of Pnc1-TT already at 1 week of age. High levels were maintained for >12 weeks. However, a PPS-1 booster induced no response in neonatal or infant mice. The adjuvant LT-K63 significantly enhanced the IgG response and affinity to Pnc1-TT by both the s.c. and the intranasal (i.n.) route in all age groups. In neonatal and infant mice, PPS-1 and LT-K63 induced a booster response only when given i.n. following either s.c. or i.n. priming with Pnc1-TT and LT-K63. In contrast, PPS-1 with or without LT-K63 administered s.c. compromised the ongoing PPS-1-specific response elicited in neonatal mice by either s.c. or i.n. priming with Pnc1-TT and LT-K63. These results demonstrate the advantage of the mucosal route for elicitation of PS-specific memory responses in early life.     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.     

Early detection of negative BACTEC MGIT 960 cultures by PCR-reverse cross-blot hybridization assay

We evaluated the efficacy of a PCR-reverse cross-blot hybridization assay, a test which permits identification of mycobacteria by means of species-specific probes and a Mycobacterium-specific probe, for early detection of negative BACTEC MGIT 960 mycobacterial cultures. Aliquots of 549 cultures were collected 7 days after the culture media were inoculated with various clinical specimens and tested with the molecular assay. PCR results were compared to those obtained at the end times with the BACTEC MGIT 960 system. Of the 549 specimens analyzed, 484 were found to be negative and 64 were found positive by both methods; one specimen, found to be positive by the BACTEC MGIT 960 system, was identified as negative by the molecular assay. In view of its high negative predictive value (99.8%), the PCR-reverse cross-blot hybridization assay appears to be a valid tool for early detection of negative BACTEC MGIT 960 cultures.     

Separate and variably shaped chromosome arm domains are disclosed by chromosome arm painting in human cell nuclei

Fluorescence in situ hybridization (FISH) with microdissection probes from human chromosomes 3 and 6 was applied to visualize arm and subregional band domains in human amniotic fluid cell nuclei. Confocal laser scanning microscopy and quantitative three-dimensional image analysis showed a pronounced variability of p- and q-arm domain arrangements and shapes. Apparent intermingling of neighbouring arm domains was limited to the domain surface. Three-dimensional distance measurements with pter and qter probes supported a high variability of chromosome territory folding.     

The effect of sympathetic stimulation on proximal brachial artery mechanics in humans--differential behaviour within the length of the brachial artery?

AIMS: The mechanical properties of arteries play a major role in the regulation of blood pressure and cardiac performance. The effect of sympathetic stimulation on the mechanical properties of the proximal brachial artery was analysed in 18 healthy volunteers, nine young (25 +/- 2 years) and nine elderly (69 +/- 2 years). METHODS: A non-invasive ultrasonic echo-tracking system for measurement of systolic/diastolic variation of the proximal brachial artery diameter in combination with intra-arterial pressure measurements was used to determine wall mechanics. The pressure-diameter (P-D) relationship, distensibility coefficient (DC), compliance coefficient (CC) and stiffness(beta) were obtained at rest and during sympathetic stimulation induced by lower body negative pressure (LBNP). RESULTS: The peripheral vascular resistance increased by 100 and 72%, respectively in the young and elderly during LBNP (P < 0.001). Simultaneously, the mechanical properties of the proximal brachial artery remained unaltered, as estimated from both P-D relationship and stiffness in young (beta-index rest: 5.2 +/- 0.9, LBNP: 5.5 +/- 1.3, NS) as well as elderly (beta-index rest: 13.6 +/- 4.6, LBNP: 16.1 +/- 4.7, NS). CONCLUSIONS: LBNP-induced sympathetic activation does not change proximal brachial artery mechanics, in contrast to earlier reports on the muscular distal brachial artery. This may imply that the transition between elastic and muscular artery behaviour is within the length of the brachial artery, where the site of transition from elastic to muscular wall structure needs to be specified in future studies.     

Cross-bridge movement and stiffness during the rise of tension in skeletal muscle – a theoretical analysis

Predictions for the time courses of cross-bridge attachment, N(t), stiffness, S(t), and force, T(t), during the tetanus rise were analysed for a special class of cross-bridge models where cross-bridges initially attach in a non-stereospecific weak-binding state, A _w. This state is in rapid equilibrium (equilibrium constant K) with detached states and the force generating transition (rate constant F ₊) is delayed. One model (model IA) which assumed step-function rise of activation at onset of tetanus, gave a poor fit to the experimental data (judged by root mean square error, RMSe 0.038) but the experimentally observed lead of N(t) over T(t) was reproduced qualitatively. An activation mechanism where K increased towards its maximum value according to an exponential function (Model IB) improved the fit considerably (RMSe 0.013). However, the activation time constant ( = 30 ms) derived in the fit was too high to reflect Ca²⁺ binding to troponin. In a further developed model (model II) both Ca²⁺-binding to troponin and cross-bridge attachment were assumed to be required for full activation. This more complex model gave a good fit to the experimental data (RMSe 0.013) with a realistic time constant for Ca²⁺ binding to troponin (9 ms). In both model IB and model II the best fit was obtained with F ₊ 40 s^–1 . An extended version of model IB, with distributed cross-bridge attachment and a series elastic element, gave a fit of similar quality (RMSe 0.009) as obtained with model IB and model II and with a similar value of F₊. The results support the view that weakly bound cross-bridges (state A _w) may account for the lead of cross-bridge movement over force during tension rise. It is also shown that, if the stiffness of the myofilaments is non-linear (stiffness increasing with tension) the experimentally observed lead of S(t) over T(t) may, to a significant degree, be attributed to cross-bridges in the state A _w.     

Characterization of the large-conductance Ca-activated K channel in myocytes of rat saphenous artery

We used the patch-clamp method to characterize the BK channel in freshly isolated myocytes from the saphenous branch of the rat femoral artery. Single-channel recordings revealed that the BK channel had a conductance of 187 pS in symmetrical 150 mM KCl, was blocked by external tetraethylammonium (TEA) with a KD(TEA) of approx. 300 microM at +40 mV, and by submicromolar charybdotoxin (CTX). The sensitivity of the BK channel to Ca was especially high (KD(ca) approx. 0.1 microM at +60 mV) compared to skeletal muscle and neuronal tissues. We also investigated the macroscopic K current, which under certain conditions is essentially sustained by BK channels. This conclusion is based on the findings that the macroscopic current activated upon depolarization follows a single exponential time course and is virtually fully blocked by 100 nM CTX and 5 mM external TEA. We made use of this occurrence to assess the voltage and Ca dependence of the macroscopic BK current. In intact myocytes, the BK channel showed a strong and voltage-dependent reduction of the outward current (62% at +40 mV), most likely due to block by intracellular Ba and polyamines. The results obtained from macroscopic and unitary current indicate that approx. 2.5% of the BK channels are active under physiological conditions, sustaining approx. 20 pA of outward current. Given the high input resistance of these cells, few BK channels are required to open in order to cause a significant membrane hyperpolarization, and thus function to limit the contraction resulting from acute increases in intravascular pressure, or in response to hypertensive pathologies.