Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence.

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.

Deficiency of the carnitine transporter (OCTN2) with partial N-acetylglutamate synthase (NAGS) deficiency.

A patient with recurrent episodes of hyperammonaemia (highest ammonia level recorded 229 micromol/L, normal 9-33) leading to altered levels of consciousness was diagnosed with partial N-acetylglutamate synthase (NAGS) deficiency (9% residual activity) at age 5 years and was treated with ammonia-conjugating agents (Ucephan 250 mg/kg per day and later sodium phenylbutyrate 200-250 mg/kg per day) for 15 years. A chronically low serum carnitine level (pretreatment plasma free carnitine 4 nmol/L, normal 37 +/- 8 nmol/L; total carnitine 8 nmol/L, normal 46 +/- 10) was assumed to be secondary and was treated with supplemental carnitine (30-50 mg/kg per day). Hypoglycaemia (blood sugar 35 mg/dl, normal 70-100), cardiomegaly, and fatty liver were also noted at diagnosis. The patient died unexpectedly at age 20 years. In retrospect, it was learned that the patient had stopped his carnitine without medical consultation several weeks prior to his death. Additional molecular investigations identified two mutations (R254X and IVS3 + 1G > A) in the patient's OCTN2 (SLC22A5) gene, consistent with a diagnosis of primary carnitine deficiency due to carnitine transporter defect. R245X is a founder mutation in Southern Chinese populations. It is unknown whether the original NAGS deficiency was primary or secondary, but molecular analysis of the NAGS gene failed to identify mutations. Urea cycle enzyme expression may be affected by fatty acid suppression of an AP-1 binding site in the promoter enhancer region of the urea cycle gene. Regardless, it is clear that the NAGS abnormality has led to delay of recognition of the OCTN2 defect, and modified the clinical course in this patient.

Treatment of Niemann-Pick disease type C in two children with miglustat: initial responses and maintenance of effects over 1 year.

Niemann-Pick disease type C (NP-C) is a lipid storage disorder characterized by the accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system of certain cells in the central nervous system (CNS) and visceral organs. Clinical symptoms include progressive neurological deterioration and visceral organomegaly. Miglustat, a small iminosugar molecule approved for the treatment of Gaucher disease, reversibly inhibits glucosylceramide synthase, which catalyses the first committed step in glycosphingolipid synthesis. The physicochemical properties of miglustat allow it to cross the blood-brain barrier and suggest possible benefits in lysosomal storage diseases affecting the CNS. Here, we present findings in two children with NP-C, aged 14 years (patient 1) and 9 years (patient 2), treated with miglustat for 1 year. Before treatment, patient 1 presented with severe difficulties in swallowing and walking, and patient 2 with problems mostly affecting communication and social interaction. Videofluoroscopic studies in patient 1 demonstrated a substantial improvement in swallowing by month 6 of treatment, and ambulation index measurements indicated improved walking. Mini Mental-State Examination (MMSE) assessments in patient 2 showed cognitive improvement by month 6, which was sustained up to month 12. Liver/spleen volume and plasma chitotriosidase activities were stabilized in both cases. There was no weight loss during treatment. Patient 1 experienced severe but self-limiting paresthesia, which was not associated with peripheral neuropathy. We conclude that miglustat can provide therapeutic benefits in CNS symptoms and allows stabilization of systemic disease in childhood-onset NP-C. Further follow-up is crucial to determine the long-term maintenance of these effects.

Gamma delta T cell receptors.

Gamma delta (gamma delta) T cells are among the least understood components of the immune system. While these cells appear to contribute uniquely to host immune competence, defining their functions in the context of host biology and pathology has been difficult. This is largely because it is unclear what antigens the gamma delta T cell receptor repertoire is directed against. During the past year, there have been noteworthy advances in this area. Their significance in the context of gamma delta T cell biology is discussed.

Identification of variations in the human phosphoinositide 3-kinase p110delta gene in children with primary B-cell immunodeficiency of unknown aetiology.

Our recent study demonstrated that defects in p110delta result in B-cell immunodeficiency that is very similar to that observed in BTK-deficient mice. We revealed that the p110delta fit the B-cell signal transduction complex and played a non-redundant role in the development and function of B cells. In humans, most children with primary B-cell immunodeficiency have mutations in the BTK, whereas a few have defects in the components of the B-cell signal transduction complex. But little is known about the genetic variation of p110delta in children with defects in B-cell immunodeficiency of unknown aetiology. Sixteen patients from 15 unrelated families and 112 normal controls underwent sequence analysis to identify genetic variations of the p110delta. Allele frequency in each group was also analysed and compared. We identified five single base-pair polymorphic nucleotide exchanges in both patient and control groups with similar allele frequencies, which did not contribute to the immunodeficiency. Three of them are novel (m.953A>G, m.1200C>T and m.1561A>G), and the m.953A>G and m.1561A>G nucleotide exchanges are non-synonymous (N253S and T456A, respectively). The novel m.1561A>G was in complete linkage disequilibrium with the known m.873A>G in our study of Taiwanese group. In addition, one novel single base-pair missense mutation, m.3256G>A (E1021K), was identified in one boy with typical clinical features of primary B-cell immunodeficiency and could not be found in either his family or the normal control population. By atomic structural analysis of the amino acid as well as the alignment comparison between species, it resulted in the replacement of the negative-charged amino acid E with the positive-charged amino acid K at codon 1021, located in the highly conservative and important catalytic functional domain. Our findings could shed light on further understanding the polymorphisms of p110delta in B-cell immunodeficiency and different populations. Moreover, the 3256G>A missense mutation raised the attention and warranted further extensive analysis to elucidate the role of p110delta in human immunodeficiency.

Calcium site mutations in cadherin: impact on adhesion and evidence of cooperativity.

This work describes quantitative force and bead aggregation measurements of the adhesion and binding mechanisms of canine E-cadherin mutants W2A, D134A, D103A, D216A, D325A, and D436A. The W2A mutation affects the formation of the N-terminal strand dimer, and the remaining mutations target calcium binding sites at the interdomain junctions. Surface force measurements show that the full ectodomain of canine E-cadherin forms two bound states that span two intermembrane gap distances. The outer bond coincides with adhesion between the N-terminal extracellular domains (EC1) and the inner bond corresponds to adhesion via extracellular domain 3 (EC3). The W2A, D103A, D134A, and D216A mutations all eliminated adhesion between the N-terminal domains, and they attenuated or nearly eliminated the inner bond. The W2A mutant, which does not destabilize the protein structure, attenuates binding via EC3, which is separated from the mutation by several hundred amino acids. This long-range effect suggests that the presence or absence of tryptophan-2 docking allosterically alters the adhesive function of distal sites on the protein. This finding appears to reconcile the multidomain binding mechanism with mutagenesis studies, which suggested that W2 is the sole binding interface. The effects of the calcium site mutations indicate that structural perturbations cooperatively impact large regions of the protein structure. However, the influence of the calcium sites on cadherin structure and function depends on their location in the protein.