Effects of Deletions in the Regulatory Domain on the Stability and Enzymatic Characteristics of Tyrosine Hydroxylase

BACKGROUND: Various vectors have been developed and tried for the delivery of tyrosine hydroxylase (TH) in order to supplement dopamine, which is severely deficient in Parkinson's disease, however, none of the protocols tried have yielded fruitful results that can be applied directly to humans. One of the problems revealed from previous trials was a short duration of expression of the delivered gene, that is, tyrosine hydroxylase. METHODS: To extend the stability and to improve the enzymatic characteristics of the protein, part of the regulatory domain was deleted via PCR technique. The cDNA for regulatory domain-deleted THs (dTH) were sub-cloned into a retroviral vector and the resulting recom-binant retrovirus was used to infect NIH-3T3. After selection, expression levels of TH were determined by Western blot analysis and the enzymatic characteristics were examined. RESULTS: The deletion increased steady state expression level of TH protein by 7-fold for d19TH (TH with amino acids #2-19 are deleted) and 3-fold for d31TH (TH with amino acids #2-31 are deleted. The elevated expression level of d19TH is likely due to the enhanced stability of the protein as determined by a treatment of cycloheximide. The activity of d19TH was also increased approximately by 3-fold but no increase of the L-dopa production was observed. However, the production of L-dopa was dramatically increased when GTP cyclohydrolase I (GTPCH I) was co-transfected suggesting that the activity of d19TH is dependent on the presence of cofactor. d19TH seem to be free of feedback inhibition at low concentration of dopamine (10 nM~1 nM) but more sensitive to the inhibition at high concentration of dopamine (10 mM). CONCLUSIONS: The deletion of 18 amino acids on the regulatory domain increases the stability of the protein, reduces the activity, and frees it from the feedback inhibi-tion by the end product.

Effects of Cofactor Tetrahydrobiopterin and Deletions on the Regulatory Domain of Tyrosine Hydroxylase on the Expression of Tyrosine Hydroxylase

BACKGROUND: For the treatment of Parkinson's disease, dopamine-producing cells or genes involved in producing dopamine or supporting neurons have been tested to replace conventional chemical therapies. Of these, tyrosine hydroxylase (TH) was the most widely used gene for the therapy. Trials using TH via various vectors yielded behavioral improvements in animal models but the effectiveness did not last long enough. As one of the approaches for solving this problem, the regulation of expression of the protein and mRNA of TH was studied. METHODS: Two approaches for a higher and/or more stable expression of TH were pursued. First, the effect of cofactor tetrahydrobiopterin (BH4) on the expression level of TH and second, the effect of deletion which enables TH protein to escape from protease attack, were examined. RESULTS: Cells producing BH4 showed an approximately 10-fold higher TH expression than cells expressing TH alone. When the in vitro modified TH was expressed in NIH-3T3, mutant THs showed elevated protein (17.5 ~68.6 fold) and mRNA (1.8 ~4.6 fold) expression levels at a steady state. CONCLUSIONS: Results suggest that an addition of BH4 has a more positive effect on mRNA expression levels than protein. However, the deletions seem to have a tremen-dous effect on the translation and/or protein stability, but a small effect on the mRNA level. (J Korean Neurol Assoc 19(5):514~519, 2001)

A Clinical Study of Ipsilateral Fracture of Femur and Tibia / 대한정형외과학회잡지

Twenty-six cases of fracture of the femur and tibia on the same leg were treated in Masan Koryo General Hospital during the period 1981 1986. We studied all of these patients, divided by four groups according to the method of treatment, retrospectively with analysis of treatment and end results. The results were as follows : 1. Twenty-two patients were male and four patients were female. Of twenty-six patients, eighteen patients were at their third and fourth decades. 2. The main cause of injuries was traffic accident; 23 patients(88%). 3. The most common concomitant injury was fracture and dislocation in other site(14 cases). The fat embolism developed in one case and one patient had died due to head injury. 4. The average healing time of fracture was 17 weeks in femur and 20 weeks in tibia in group three, that was most rapid of all groups. 5. A good or excellent functional result was achieved in 87% of the patients treated with internal fixation of both fracture, 75% of the patients with internal fixstion for the femur fracture and external fixation for the tibial fracture, and 25% of the patients trested conservatively.

Metastatic Retinoblastoma to Tibia: A Case Report / 대한정형외과학회잡지

Retinoblastoma is a malignant tumor that arises from the retina. The prognosis is very unfavorable if the choroid is affected or the central nervous system is invaded or if metastasis have occurred. We experienced a case of the retinoblastoma with optic nerve involvement in a 3-years-old female had metastasis to the tibia. She was enucleated of the left eye five months ago. We report it with brief review of the literature.

Apert Syndrome: A Report of One Case / 대한정형외과학회잡지

Apert described acrocephalosyndactly as a clinical entity in 1906. The classic description of this syndrome includes patient with a combination of acrocephaly and syndactyly of either fingers, toes or both. More than 200 cases have been reported in the world literature. Authors experienced on case of the classic pattern of Apert syndrome and treated surgically for syndactyly. This one case was reported on this paper with brief review of the relevant literatures.

The Treatment of Acromioclavicular separation / 대한정형외과학회잡지

There are many procedures for treatment of injuries of acromioclavicular separation, but still controversies concerning the best management of these injuries. From March 1981 to August 1984 at Masan Korea Hospital, twelve cases were treated by technique of modified Phemister method, two cases by Dewar and Barrington method, two cases by Stewart method, one case by Neviaser method, three cases by skillful neglect method and two cases by shoulder harness immobilization. The following results were obtained. 1. These injuries were more prevalent in male and more in right side. 2. The most common cause of injuries was traffic accident and followed by falling from the height. 3. Three cases were grade 2 and nineteen cases grade 3 by Allman's classification. 4. We treated these injuries by operative method in seventeen cases and non-operative method in five cases. 5. There were good functional results of treatment in operative method. 6. There were two cases of superficial wound infection, two cases of migration of K-wire and one case of recurrence of deformity in operative method, and two cases of shoulder stiffness and 2 cases of recurrence of deformity in non-operative method.

The Clinical Observation of the Femoral Shaft Fractures in Children / 대한정형외과학회잡지

The managements of fractures of femoral shaft in children differ in many ways from those in adults. Fourty eight cases of fractures of femoral shaft in children who were treated at Masan Korea General Hospital from March 1981 to February 1984 were analyzed both clinically and radiologically and the following results were obtained. l. Among 48 cases, the ratio of male and female was about 2.7:1 and the highest incidence was between the age of 3 and 8 years(52.1%). 2. The main cause of fracture was traffic accident(66.7%) and the other was falling or slip down injury. 3. Middle 1/3 of femoral shaft was most commonly injured(45.8%) and transverse type was common (41.7 %). 4. The common associated injuries were head injuries, fracture of the lower extremities and fracture of pelvis. 5. 43 cases were treated with traction method and 5 with open reduction and internal fixation. 6. In general, angulation at fracture site was under 10 degrees and no clinical deformity was resulted in. 7. Among 48 cases, overring of fractured fragment was occured in 28 cases and the average length of overring was 7.5mm.

A Study on Relation betwen Red Cell Electrolyte Concentractions and Serum Digoxin Level

For the purpose of elucidating relation between red cell electrolyte concentrations and serum digoxin level, measurement of red cell electrolyte concentrations and seum digoxin level by radioimmunoassay were done in 46 normal controls and 63 patients of CHF including 8 patients with digitalis intoxication. The results obtained were as follows: 1. Red cell sodium concentration and ratio of red cell sodium to red cell potassium concentration in normal males were significantly higher than those of females, and red cell potassium concentration in normal males was significantly lower than that of normal females. 2. Red cell potassium concentration in patients of CHF was significantly higher than that of normal controls, but there was no difference in the red cell sodium concentration between the groups. 3. There was no relation between red cell electrolyte concentrations and serum digoxin level, and red cell electrolyte concentration did not exactly estimate serum digoxin level. 4. Red cell electrolyte concentrations in the patient of CHF were valuable in detecting digitalis toxicity.

Progressive Muscular Dystrophy (Report of 32 cases) / 대한정형외과학회잡지

Progressive muscular dystrophy is a genetically determined myopathy of unknown etiology and is a primary degenerative disease of skeletal muscle fibers. The authors reviewed 32 cases of progressive muscular dystrophy and made following observations. 1. The types among the 32 cases were as follows. 1) Duchenne type ; 16 2) Limb-Girdle type ;12 3) Facioscapulohumeral type ;3 4) Distal myopathy ;1 5) Ocular myopathy ;0 2. 13 of the 32 patients had relevant family history. 3. In general, creatinine excretion was decreased in 24-hour urine and creatine was increased. 4. 6 of the 10 patients tested had increased serum C.P.K. level.

An epidemiologic Study of Tennis Elbow in Tennis Player / 대한정형외과학회잡지

The etiologic factors of tennis elbow have been obscure in spite of increased concern and study. The true pathologic changes in tennis elbow are also not well established. The authors anslized 300 tennis players for a history of tennis elbow, finger size, body weight,weight and grip size of racket etc. The results were as follows; 1. 68 among 300 players have suffered from tennis elbow more than one time (22.7%). 2. There were no difference in correlation between body weight and racket weight. 3. Most players have used improper grip, especially in those who have suffered from tennis elbow.

Two cases of Benign Mesenchymoma arising in Thigh and Forearm / 대한정형외과학회잡지

The criteria for a mesenchymoma are that it should be composed of at least two mesenchymal elements not ordinarily found together in a tumor. This report dealt with two cases of benign mesenchymoma arising, one in the left thigh and the other in the left forearm. To diagnose of this tumor on X-ray preoperatively was difficult. The tumors were excised as much as possible, and mesenchymal derivatives characteristic of the tumor was described in detail.