Hypervariable tandem repetitive regions in human DNA are proving to be increasingly useful for genetic analysis in humans. We chose four single locus probes (SLP; MS1, MS43, MS8 and g3) for a validation test among Koreans. The specimens were from 216 unrelated individuals and 33 paternity inclusion families. Extracted DNA from EDTA blood was restricted by Hinfl and electrophoresed in 0.7% agarose gel, transferred and hybridized with chemiluminescent probes. Heterozygosity was over 90% by all of the probes. Total numbers of unassignable mutant bands from 33 paternity inclusion cases were 5, and the highest mutation rate was determined in probe MS1(0.045). The probability of having the same DNA band between two unrelated individuals was 5.7 x 10(-10) when four SLPs were used at the same time. The data presented here on allele frequencies and mutation rates provide preliminary data supporting the validity of these probes in paternity analysis and forensic investigators in the Korean population.
Alleles ; Chromosome Mapping ; DNA, Satellite/*genetics ; Female ; Heterozygote ; Human ; Male ; Mutation ; Myoglobin/*genetics ; *Paternity ; Support, Non-U.S. Gov't

BACKGROUNDMyoglobin is expressed exclusively in striated skeletal muscles and has been implicated in nitric oxide scavenging. Accumulating data suggest a critical role for nitric oxide in both the endogenous and therapeutic angiogenic response to ischemia. A clear role for myoglobin in ischemic skeletal muscle is uncertain. We hypothesized that myoglobin overexpression has an adverse impact on the angiogenic response to ischemia.

METHODSMuscle-specific myoglobin over-expressing transgenic mice (MbTG, n = 11), wild type littermates (WT, n = 23) underwent unilateral femoral artery ligation and excision. Laser doppler perfusion imaging was used to monitor changes in hindlimb perfusion before surgery and weekly after surgery up to 28 days. Tissue ischemia was assessed by a necrosis incidence. Upon termination of the experiment (28 days after surgery), skeletal muscles (gastrocnemius, and tibialis anterior) were harvested, the distal part of the muscle was frozen and embedded for histology study, the proximal part was used either to detect vascular endothelial growth factor (VEGF) level with enzyme-linked immunosorbent assays (ELISA) or to determine the proliferation (proliferating cell nuclear antigen (PCNA)) and apoptosis (Bax, and Bcl-2) condition in ischemic muscle by Western blotting. Capillaries were stained with endothelial phosphate alkaline staining and vascular density was expressed in capillaries/fiber.

RESULTSThe recovery of perfusion in MbTG mice was similar to that of WT mice on day 7 (0.485 +/- 0.095 vs 0.500 +/- 0.084) but was significantly less on day 14 (0.536 +/- 0.086 vs 0.623 +/- 0.077, P < 0.05), day 21 (0.588 +/- 0.082 vs 0.684 +/- 0.068, P < 0.01) and day 28 (0.606 +/- 0.079 vs 0.733 +/- 0.093, P < 0.01). The necrosis incidence was higher in MbTG than in WT (54.5% vs 21.6%). Vascular density was less in MbTG compared with that in WT (gastrocnemius 0.19 +/- 0.08 vs 0.30 +/- 0.08, P < 0.05; tibialis anterior 0.22 +/- 0.11 vs 0.33 +/- 0.04, P < 0.05). With ischemic injury, the VEGF level was increased in both MbTG and WT (45.2% and 20.4%, respectively). Western blotting showed that after hindlimb ischemia the proliferation was similar in both MbTG and WT, however, apoptosis was increased in MbTG relative to WT, shown as more expression of Bax and less expression of Bcl-2.

CONCLUSIONAn increase in expression of myoglobin protein in skeletal muscle reduces the endogenous perfusion recovery following surgically induced hind-limb ischemia.

Animals ; Apoptosis ; genetics ; Blotting, Western ; Cell Proliferation ; Enzyme-Linked Immunosorbent Assay ; Hindlimb ; blood supply ; metabolism ; Immunohistochemistry ; Ischemia ; therapy ; Mice ; Mice, Transgenic ; Muscle, Skeletal ; blood supply ; cytology ; metabolism ; Myoglobin ; genetics ; physiology

OBJECTIVETo explore the application of caffeine-halothane contracture test (CHCT) in the confirmation of malignant hyperthermia (MH).

METHODSOne patient who underwent radical gastrectomy presented with clinical manifestations of MH during routine intravenous-inhalation anesthesia process. Isoflurane inhalation and the operation were ceased immediately and emergency management approaches such as physical cooling therapy were taken. Meanwhile, the levels of serum creatine kinase (CK), serum myoglobin, and urinary myoglobin were examined and rectus abdominis was taken and then CHCT was performed to confirm the clinical diagnosis. Total genome was extracted from the patient and then exons 2-18, 39-46, and 90-104 of ryanodine receptor 1 (RYR1) gene were screened to detect mutations using DNA sequencing technique.

RESULTSThe patient was diagnosed as MH episode by clinical characteristics and postoperatively continuous elevation of the levels of CK, serum myoglobin, and urinary myoglobin (30 times higher than normal level). Despite halothane test was negative, the diagnosis of MH was verified by the positive result of caffeine test. DNA sequencing of RYR1 gene of the patient revealed c. 6724C > T (p. T 2 206M).

CONCLUSIONCHCT can be used to confirm the diagnosis of MH.

Anesthetics, Inhalation ; therapeutic use ; Caffeine ; Creatine Kinase ; blood ; Enzyme-Linked Immunosorbent Assay ; Halothane ; Humans ; Isoflurane ; therapeutic use ; Malignant Hyperthermia ; blood ; diagnosis ; genetics ; Muscle, Skeletal ; drug effects ; physiopathology ; Myoglobin ; blood ; Ryanodine Receptor Calcium Release Channel ; genetics