Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiency

An infant girl with elevated blood lactate, pyruvate, and plasma branched-chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3; dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) deficiency. Activities of the pyruvate dehydrogenase complex and E3 from patient were 26 and 2% of controls in blood lymphocytes, and 11 and 14% in cultured skin fibroblasts, respectively. Western blot analysis demonstrated that the amount of E3 protein in fibroblasts from the patient and her father was about half of controls, while Northern blot analysis showed normal amounts of E3 RNA. DNA sequencing of cloned full-length E3 cDNAs from the patient revealed two mutations in separate alleles. One is a single base insertion of an extra adenine in the last codon of the leader peptide sequence (TAC-->TAAC) leading to a nonsense mutation which results in the premature termination of the precursor E3 polypeptide (Y35X). The other is a missense mutation due to substitution of guanine for adenine, causing an Arg-->Gly substitution at amino acid 460 of the mature protein (R460G) which triggers the loss of E3 activity probably by structural change in the E3 dimer. DNA sequencing of E3 cDNAs from the parents demonstrated that the nonsense mutation was inherited from the father and the missense mutation was inherited from the mother.      

Two color flow cytometry of lymphocytes from patients with adult T-cell leukemia

Lymphocytes from eight patients with adult T-cell leukemia were analyzed by two color flow cytometry. Monoclonal antibodies (Leu 3 a, Leu 8, Leu 2 a and Leu 15) labelled with fluorescein isothiocyanate or phycoerythrin were used. The purpose was to identify the subsets of the lymphocytes as helper, suppressor/inducer, suppressor or cytotoxic by the surface marker of the cells. All eight patients had antibodies for ATLA. Proviral DNA in the lymphocytes was found in six patients. Summarising the results, OKT4-positive ATL cells were all of the helper T-cell subset, not the inducer subset. OKT8-positive ATL cells were also positive for OKT4 and were all of the cytotoxic T cell subset, not the suppressor subset. In two patients, some ATL cells had both OKT4 and OKT8 on the same cells, especially in the lymph nodes. In our study, ATL cells from eight cases of ATL had all of the helper T subset. These results suggest that the target cells of the human T cell leukemia/lymphoma virus type will be helper T cells.     

Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection

The present study evaluated the potential use of immunoglobulin prepared from the egg yolk of hens immunized with Helicobacter pylori (immunoglobulin Y [IgY]-Hp) in the treatment of H. pylori infections. The purity of our purified IgY-Hp was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk. The titer for IgY-Hp was 16 times higher than that for IgY in egg yolk from nonimmunized hens, and IgY-Hp significantly inhibited the growth and urease activity of H. pylori in vitro. Bacterial adhesion on AGS cells was definitely reduced by preincubation of both H. pylori (10(8) CFU/ml) and 10 mg of IgY-Hp/ml. In Mongolian gerbil models, IgY-Hp decreased H. pylori-induced gastric mucosal injury as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, in this experimental model, H. pylori-associated gastritis could be successfully treated by orally administered IgY-Hp. The immunological activity of IgY-Hp stayed active at 60 degrees C for 10 min, suggesting that pasteurization can be applied to sterilize the product. Fortification of food products with this immunoglobulin would significantly decrease the H. pylori infection. In conclusion, the IgY-Hp obtained from hens immunized by H. pylori could provide a novel alternative approach to treatment of H. pylori infection.     

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

Leg-length discrepancy and associated risk factors after paediatric femur shaft fracture: a multicentre study

PurposeThis study was performed to investigate leg-length discrepancy (LLD) and associated risk factors after paediatric femur shaft fractures.MethodsA total of 72 consecutive patients under 13 years old (mean age 6.7 years; 48 boys, 24 girls) with unilateral femur shaft fracture, and a minimum follow-up of 18 months, were included. The amount of LLD was calculated by subtracting the length of the uninjured from that of the injured limb. Risk factors for an LLD ≥ 1 cm and ≥ 2 cm were analyzed using multivariable logistic regression analysis.ResultsHip spica casting, titanium elastic nailing and plating were performed on 22, 40 and ten patients, respectively. The mean LLD was 7.8 mm (sd 8.8) and 29 (40.3%) had a LLD of ≥ 1 cm, while nine (12.5%) had a LLD of ≥ 2 cm. There were significant differences in fracture stability (p = 0.005) and treatment methods (p = 0.011) between patients with LLD < 1 cm and ≥ 1 cm. There were significant differences in fracture site shortening (p < 0.001) and LLD (p < 0.001) between patients with length-stable and length-unstable fractures. Fracture stability was the only factor associated with LLD ≥ 1 cm (odds ratio of 4.0; p = 0.020) in the multivariable analysis.ConclusionThis study demonstrated that fracture stability was significantly associated with LLD after paediatric femur shaft fractures. Therefore, the surgeon should consider the possibility of LLD after length-stable femur shaft fracture in children.Level of EvidencePrognostic level III     

A novel mis-sense mutation (G1381A) in the G6PD gene identified in a Chinese man

Objective　To detect new mutations among 29 glucose-6-phosphate dehydrogenase (G6PD) deficient individuals from Yunnan province. Methods　The nitroblue tetrazolium (NBT) method was used to screen G6PD deficient individuals. Mutation was identified by single strand conformation polymorphism (SSCP), amplification created restriction site (ACRS), amplification refractory mutation system (ARMS) and DNA sequencing. Results　Among 29 cases, 18 cases of G1388A, 1 case of C1004A, and 1 case of G1381A were identified. Nine cases remained to be defined. The G1381A mutation is a novel mis-sense mutation, with a substitution of threonine for alanine (A461T). The resultant G6PD had reduced enzymatic activity. In addition, G1381A caused a restriction site of Stu I to disappear, providing a rapid method for the detection of this mutation. Conclusion　A novel mis-sense mutation G1381A was found. This mutation results in a substitution of threonine for alanine, producing enzyme with reduced activity. The loss of the Stu I restriction site offers a rapid method for the detection of this mutation.     

1H-NMR and NOE studies of 2-cephems and 3-cephems

The¹H-NMR signals of 2-cephems and 3-cephems have been assigned and the Nuclear Overhauser Effect (NOE) study of these compounds was undertaken.     

Differential pharmacology between the guinea-pig and the gorilla 5-HT1D receptor as probed with isochromans (5-HT1D-selective ligands).

1. Both the 5-HT1D and 5-HT1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5-HT1D receptors with much higher affinity than 5-HT1B receptors. In the guinea-pig, a primary animal model for anti-migraine drug testing, however, isochromans bound the 5-HT1D receptor with lower affinity than the gorilla receptor. 2. This species-specific pharmacology was investigated, using site-directed mutagenesis on cloned guinea-pig receptors heterologously expressed in human embryonic kidney 293 cells. Mutations of threonine 100 and arginine 102 at the extracellular side of transmembrane II of the guinea-pig 5-HT1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its affinity for isochromans to that of the gorilla receptor, with little effects on its affinities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. 3. For G protein-coupling, serotonin marginally enhanced GTPgamma35S binding in membranes expressing the guinea-pig 5-HT1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPgamma35S binding in the latter nearly as much as serotonin, and several isochromans by 30-60% of serotonin. 4. We discovered key differences in the function and binding properties of guinea-pig and gorilla 5-HT1D receptors, and identified contributions of I100 and H102 of primate 5-HT1D receptors to isochroman binding. Among common experimental animals, only the rabbit shares I100 and H102 with primates, and could be useful for studying isochroman actions in vivo.     

Agonist-induced GTPgamma35S binding mediated by human 5-HT(2C) receptors expressed in human embryonic kidney 293 cells

The 5-HT(2C) receptor as heterologously expressed in various mammalian cells mediates inositol 1,4,5-triphosphate (IP(3)) signal by activating G(q/11) subtypes of G proteins, but minimally promotes agonist-induced GTPgamma35S binding in membranes due to slow GTP turnover rates of the G proteins. Here we discovered robust (over 200%) agonist-induced GTPgamma35S binding mediated by the human receptor expressed in human embryonic kidney (HEK) 293 cells, and investigated its pharmacology. Agonists concentration-dependently increased GTPgamma35S binding in isolated membranes, which was competitively blocked by antagonists. Intrinsic efficacies of agonists from GTPgamma35S binding were comparable to those from IP(3) measurement. Pertussis toxin treatment largely blocked serotonin-induced GTPgamma35S binding, serotonin high affinity sites by 70% without altering the total binding sites, and reduced IP(3) release by 40%. GTPgamma35S-bound Galpha subunits from serotonin-activated membranes were mainly Galpha(i), judging from immobilization studies with various Galpha-specific antibodies. Inhibition of forskolin-stimulated cAMP formation, however, was not observed. Apparently, the 5-HT(2C) receptor-mediated GTPgamma35S binding is a unique phenotype observed in HEK293 cells, reflecting its coupling to pertussis toxin-sensitive G(i) subtypes, which contribute to the IP(3) signal, along with pertussis toxin-insensitive G(q/11) subtypes.