Insertion/deletion mutations of type I oculocutaneous albinism in chinese patients from Taiwan

Type I oculocutaneous albinism (OCA1) is an autosomal recessive disorder, which is caused by the reduction or the absence of tyrosinase activity in melanocytes of the skin, hair and eyes. Although tyrosinase mutations of OCA1 have been extensively analyzed in most populations worldwide, there is no systemic study of OCA1 mutation in Chinese patients. By use of single strand conformation polymorphism and direct sequencing, we had detected 21 mutant alleles out of 24 OCA1 chromosomes screened (87.5%). Detected mutant alleles include one splicing site, three insertion/deletion and five missense mutations, of which the splicing site nucleotide alteration (IVS 1-3C>G) and two each of the insertion/deletion (232-233 ins GGG and 861-862 del TT) and missense mutations (Cys 289 Gly and Trp 400 Leu) are novel. The ins/del mutations accounts for about 37.5% in Chinese OCA1 alleles. The 232-233 ins GGG, one of the novel mutations, was found to be most frequent (25%) among the OCA1 alleles in Chinese. Through this study, we found that while some of the OCA mutant alleles were identified in other populations, ethnic difference still exists. Hum Mutat 14:542, 1999.     

Manganese Superoxide Dismutase Gene Polymorphisms in Psoriatic Arthritis

The purpose of the present study is to investigate the role of manganese superoxide dismutase (MnSOD) gene polymorphisms in the susceptibility to psoriatic arthritis. MnSOD gene polymorphisms were determined by polymerase chain reaction/restriction fragment length polymorphisms method in fifty-two patients with psoriatic arthritis and 90 healthy controls. The genotype frequency of MnSOD 1183C/T was significantly higher in patients with psoriatic arthritis than in controls. In contrast, the frequency of MnSOD 1183T/T was significantly decreased in patients with psoriatic arthritis. The phenotype frequency of MnSOD 1183C was significantly increased in patients with psoriatics arthritis in comparison to healthy controls. Therefore, MnSOD 1183C polymorphisms may be a precipitating factor for the development of psoriatic arthritis.     

Fatal fat embolism following amphotericin B lipid complex injection

A case of amphotericin B lipid complex induced fatal fat embolism is described. A 41-year-old Caucasian man with AIDS was undergoing treatment for cryptococcal meningitis with amphotericin B. His course was complicated by renal failure necessitating a change in therapy to amphotericin B lipid complex (Abelcet). At approximately 48 h, the patient developed tachycardia, tachypnea, respiratory failure, decline in hematocrit, thrombocytopenia, and alteration in mental status. Autopsy findings included fat emboli involving heart, lungs, kidney, and brain. To our knowledge, this is the first case report of a fatal fat embolism caused by intravenous liposome drug delivery.     

Nucleotide sequences of novel HLA-B35 subtypes

In recent reports we described novel hybridization patterns (HP) corresponding to 22 potentially new HLA-B locus alleles in a panel of 547 subjects studied by PCR-SSOP. Three of them correspond to new subtypes of B35. To confirm the hybridization results we have isolated DNA from PBL and performed PCR, DNA cloning and nucleotide sequencing. One of the alleles, locally called B-3505v was found in three individuals: two Hispanic, one Caucasoid. It differs from B^*3505 by 3 nucleotide substitutions that lead to changes in residues 94 (Ile > Thr), 95 (Ile > Leu) and 103 (Val > Leu). B-3505v differs from B^*3501 in residues 97 and 103. Another allele called B-3508v, was found in 7 individuals, (6 of 122 Toba Indians, 1 of 18 Pilaga Indians). It differs from B^*3509 in two silent nucleotide substitutions (codons 135 and 138) and in one substitution in residue 156 (Arg > Leu). The new allele has a hybrid sequence between B^* 3508 and B^*4801. A third subtype, locally called B-3504v, observed in two Hispanic individuals, is identical to B^*3512. B^*3512 differs from B^*3504 by 3 nucleotides and one amino acid. Substitutions in residue 95 contribute to the structure of specificity pocket F, 97 to C and E, and 156 to pockets D and E. Therefore it is possible that some of the new alleles may have different peptide binding profiles. Since differences in residue 156 have been shown to affect allorecognition and mediate GvHD, identification of such variants may have important implications in transplantation and perhaps in studies of immune responses to peptides and pathogens.     

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.     

Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization

The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500- to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD(50)s) of A1 toxin but less than 500 LD(50)s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies.     

Improvement of Schwann cell attachment and proliferation on modified hyaluronic acid strands by polylysine

Hyaluronic acid (HyA) has the intrinsic ability to promote cell proliferation and reduce scar formation. However, the clinical use of HyA has so far been limited because of its water solubility and nonadhesive characteristics. Increasing interest in HyA as a clinically useful biomaterial has prompted our study of altering HyA's physical properties to render it a potential component of nerve grafts. In this study, strands of HyA were cross-linked by glutaraldehyde (Glut), coated with polylysine, and then inoculated with Schwann cells (SCs). Results in vivo and in vitro demonstrated that cross-linked HyA strands were water insoluble and thus less biodegradable. Poly-D-lysine-resurfaced strands showed significant SC attachment of 350-400 cells/mm(2), compared to uncoated controls (0-10 cells/mm(2), p < 0.01). Fibroblast control groups showed an attachment of 40-100 cells/mm(2) on coated strands. Immunostaining for proliferating cells showed SCs as and fibroblasts as +. Cells neither adhered to nor proliferated on the modified HyA strands that were not resurfaced. The results suggest that polylysine promotes SC attachment and proliferation to glutaraldehyde-cross-linked HyA strands, the product being a three-dimensional composite with low solubility that may have potential application in nerve grafts.     

Alterations of plasma antioxidants and mitochondrial DNA mutation in hair follicles of smokers

The effects of long-term smoking on mitochondrial DNA (mtDNA) deletions in hair follicles were investigated in subjects with different antioxidant capacity. Twenty-two male smokers with a smoking index of greater than 5 pack-years and without any known systemic diseases were recruited for this study. Forty healthy nonsmoking males were included as controls. We found that the concentrations of ascorbate and alpha-tocopherol and the activities of glutathione S-transferase (GST) and glutathione peroxidase in blood plasma were significantly decreased in smokers. The levels of glutathione and protein thiols in whole blood and the incidence of a 4,977 bp deletion of mtDNA (dmtDNA) in hair follicles were significantly increased in smokers. A significantly higher incidence of the 4,977 bp dmtDNA was found in smokers with plasma GST activity less than 5.66 U/l (OR = 7.2, P = 0.020). Using multiple covariate ANOVA and logistic regression, we found that age and low plasma GST activity were the only two risk factors for the 4,977 bp dmtDNA. These results suggest that smoking depletes antioxidants and causes mtDNA deletions and that plasma GST may play an important role in the preservation of the mitochondrial genome in tissue cells of smokers.