ADA Enzyme Research Articles

Successful Peripheral T-Lymphocyte–Directed Gene Transfer for a Patient With Severe Combined Immune Deficiency Caused by Adenosine Deaminase Deficiency

AbstractTen patients with adenosine deaminase deficiency (ADA−) have been enrolled in gene therapy clinical trials since the first patient was treated in September 1990. We describe a Japanese ADA− severe combined immune deficiency (SCID) patient who has received periodic infusions of genetically modified autologous T lymphocytes transduced with the human ADA cDNA containing retroviral vector LASN. The percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% during the 12 months since the fourth infusion. ADA enzyme activity in the patient's circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual and was associated with increased T-lymphocyte counts and improvement of the patient's immune function. The results obtained in this trial are in agreement with previously published observations and support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA−SCID.

Plant Regeneration of Hot Pepper (Capsicum annuum L.) and Expression of Mouse Adenosine Deaminase (ADA) Gene via Agrobacterium-mediated Transformation

Since in vitro regeneration and transformation systems in hot pepper (Capsicum annuum L.) have not been available, the application of new genetic manipulations has been limited. Here we report an efficient procedure to regenerate whole pepper plants and to generate transgenic plants expressing a foreign gene was established. High frequency of plant regeneration was observed when hypocotyl and cotyledon explants were cultured on MS/B5 medium supplemented with NAA 0.05 mg·L–1 plus zeatin 2.0 mg·L–1, NAA 0.05 mg·L–1 plus zeatin 2.0 mg·L–1, IBA 10.0 mg·L–1 plus BA 1.0 mg·L–1, IAA 0.02 mg·L–1 plus zeatin 3.0 mg·L–1. An addition of AgNO3 5–10 μm to these media improved the regeneration rate by about 10%. For plant transformation, hypocotyl and cotyledon explants of pepper were preconditioned on kanamycin-free shoot induction medium for 48 hours. Then, co-cultivation with Agrobacterium tumeaacience was done on the co-culture medium for 2 days. The explants were then blotted in sterile filter paper and placed on shoot induction and selection medium containing kanamycin sulfate (100 mg·L–1) and carbenicillin (500 mg·L–1). PCR showed that the introduced ADA gene was integrated and stably expressed in the regenerated plants. ADA enzyme activities were checked by spectrophotometric analysis.

Genetic heterogeneity and population structure in eastern India: Red cell enzyme variability in ten Assamese populations

This paper is a part of the genetic study of the people of Assam (eastern India), initiated by the Anthropometry and Human Genetics Unit, Indian Statistical Institute, Calcutta, India, and the Dept. of Human Biology/Physical Anthropology, University of Bremen, W. Germany. The results of 1. allele distribution of five red cell enzyme polymorphisms in ten Assamese populations, 2. heterogeneity of allele frequencies and extent of gene differentiation among these populations, and 3. standard genetic distances are presented here. A total of 1024 blood samples was screened for aP, E D, AK, ADA and LDH enzyme systems for Brahmins, Kalitas, Kaibartas, Rajbanshis, Muslims, Ahoms, Chutiyas, Kacharis, Karbis (Mikirs) and Sonowals, of which the latter three are tribes. The gene diversity (FST) is smallest (0.0035) for pa and highest (0.1604) for HbE. The total FST value (0.0399 +/- 0.0141) appears to be statistically significant. From distance analysis two major clusters with sub-clusters in each are visible, which are in conformity with the ethnohistory of these populations.

14 LONG TERM EXPRESSION OF HUMAN ADENOSINE DEAMINASE IN MURINE HEMATOPOIETIC CELLS

Twenty-two defective retrovirus vectors carrying human adenosine (ADA) coding sequences were tested for efficient transduction of human ADA enzyme activity. These vectors were designed to test functional aspects of construction including the use of internal transcriptional units, the importance of the Moloney sequences 563-1038 (gag+), and alterations in the 3′ LTR. Promoters from the human ADA and cFos genes, herpes virus thymidine kinase, and cytomegalovirus immediate early region gene were tested as alternatives to the viral LTR promoter. Several vectors all containing the gag+ region allow high titer virus production. These vectors were then tested in bone marrow transplant experiments. Two vectors, one utilizing the Moloney LTR promoter and one with the HSVTK promoter, gave expression of ADA in CFU-C, CFU-S and in the blood of reconstituted animals. Infection efficiency ranged from 15-85% in CFU-S by Southern analysis. Expression efficiency was also variable. All animals had human ADA in blood 2-4 weeks post-transplant but at 10-12 weeks levels of the enzyme declined. Four of 14 long term recipients however, demonstrated expression for up to 5 months. PCR analysis on nonexpressors indicated a loss of cell with the proviral DNA suggesting that the initial expression resulted from infection of more mature progenitors. Immuno-staining is being used to characterize the lineages and distribution of ADA producing cells in the transplant recipients.

Inhibition of 3H-thymidine incorporation by adenosine and deoxyadenosine in human peripheral lymphocytes and malignant lymphoid cell lines.

The purine converting enzyme adenosine deaminase (ADA, E.C. 3.5.4.4.) catalyzes the conversion of adenosine and deoxyadenosine into inosine and deoxyinosine respectively. The inherited deficiency of ADA is associated with an impairment of both cellular and humoral immunity1,2. Although all cells of patients with ADA deficiency lack a functional ADA enzyme, the expression of the deficiency seems to be restricted mainly to the immune system. This selective effect of ADA deficiency on lymphoid cells has drawn attention to the possibility of using ADA inhibitors as potential selective chemotherapeutic agents in treatment of lymphoproliferative disorders3.

Red cell antigen, serum protein, and red cell enzyme polymorphisms in inhabitants of the Jimi Valley, Western Highlands, New Guinea.

A series of blood samples from four villages in the Jimi Valley, Western New Guinea Highlands, has been tested for genetic variation in blood group, serum protein, and red cell enzyme systems. Polymorphic variation was present for the AB0, MNS, P, and Rh blood group systems, for the Hp and Tf serum protein systems, and for the acid phosphatase, 6-PGD, PGM, MDH, and ADA enzyme systems. One each of the following variants was detected: Ge(a-), G6PD deficient, AK 2-1 and PHI 7-1 or 8-1. All samples tested were Cw-, K-, Kp(a-), Wr(a-), Fy(a+ b-), Rd-, and LDH normal. Genetic distance analysis places the Jimi Valley populations closer to peoples of the Chimbu-Chuave and Wahgi-Hagen areas than to the Maring people of the Simbai Valley to the north.

Plasma protein and enzyme polymorphisms in Belgium.

We report the frequencies of the Hp, Tf, Gc, Gm, Km, Ag and C3 plasma groups, and of the AcP1, PGD, PGM1, AK1, ADA, s-GPT and EsD enzyme groups, and we list the rare variants we observed in a Belgian population samples from the Liège region.

豚の赤血球酵素PHI,6PGD,PGMおよびADA型の出現頻度の品種間差異と親子鑑別における有効性

日本のランドレース(L),ハンプシャー(H),大ョークシャー(W),デュロック(D)種の4豚品種集団から任意に抽出された464頭の赤血球酵素PHI,6PGD,PGM,ADA型を調査し,酵素型の出現頻度の品種間比較を行った.そして遺伝子頻度を基にして,各酵素型の親子鑑別における有効性を検討した.PHI,6PGD,PGM, ADA酵素はそれぞれ調査したすべての品種で多型を示し,既知の遺伝子が各々の酵素システムで観察された.PHIシステムではPHIAとPHIB遺伝子が見出され,すべての品種においてPHIBの頻度がPHIAより高かった.6PGDシステムでは6PGDAと6PGDB遺伝子が見出され,品種間に著しい出現頻度の差異がみられた.すなわち6PGDAの頻度はHとW種では6PGDBよりかにり高かったが,逆にD種ではかにり低かった.PGMシステムではPGMAとPGMB遺伝子が見出され,PGMBの頻度はPGMAよりすべての品種におい内高く,L種以外の品種では大きに差異を示した. ADAシステムでは3つの遺伝子(ADAA, ADAB, ADAO)が見出され,その出現頻度は品種間で著しい差異を示した.LとW種では3つの遺伝子が存在していたが,H種ではADABが,D種ではADAOが見出されなかった.D,WおよびL種ではADAAが圧倒的に高い頻度を示したが,H種ではADAOの頻度が圧倒的に高かった.次にこれらの酵素型座位による父権否定の確率を各品種ごとに求めると,4っの座位を組み合せた確率として,L:0.482,H:0.214,W:0.326,D:0.329の値が得られた.そして赤血球抗原型7座位,血清蛋白質型5座位,血清アロタイプ2座位と組み合せた合計18座位によって父権否定の確率は,L:0.926,H:1.829,W:0.883,D:0.814の値に達した.

Conversion of human erythrocyte-adenosine deaminase activity to different tissue-specific isozymes. Evidence for a common catalytic unit.

Adenosine deaminase activity resides in various characteristic isozymes in red blood cells (RBC-ADA) and other tissues. Absence of RBC-ADA has been reported in a proportion of patients with autosomally inherited severe combined immunodeficiency (SCID). We have previously reported that the tissue isozymes of ADA are also deficient in children with SCID and RBC-ADA deficiency, although these isozymes differ from RBC-ADA in molecular weight, accessible SH groups, and electrophoretic mobility. The deficiency of all types of ADA in SCID implies that a catalytic unit of ADA in each isozyme is coded by the same structural gene. The relationship of RBC-ADA and the different tissue ADA isozymes is the subject of this paper. Incubation of RBC-ADA with ADA-deficient liver, kidney, and fibroblast extracts resulted in the appearance of new isozymes of ADA. These newly generated isozymes had the physicochemical and electrophoretic characteristics of the tissue-specific isozymes obtained from normal tissues. The electrophoretic mobility of the isozyme generated appeared to depend upon the tissue utilized and corresponded to the electrophoretic mobilities of the ADA isozymes found naturally in each of the different tissues. Additionally, the genetically determined polymorphism exhibited by RBC-ADA could be detected in the isozyme generated. Incubation with normal kidney also caused conversion of the RBC isozyme to the kidney form. These findings further support the concept that the catalytic activity of each of the several forms of the ADA enzyme resides in a single molecule coded at the same genetic locus as is defective in one form of SCID. The tissue-specific isozymes, which differ in electrophoretic mobility and molecular weight, are generated by interaction of the RBC catalytic unit with tissue-specific factors present in the different tissues of normal humans and patients.

The distribution of serum protein and enzyme groups among the batak of Samosir Island (Sumatra, Indonesia).

188 blood samples from Batak of Samosir Island (Sumatra, Indonesia) have been studied for electrophoretic variants of haemoglobin, 14 red cell enzyme and 5 serum protein systems. The acid phosphatase, 6 PGD, PGM1 and ADA enzyme systems are polymorphic, and a single AK 2-1 person was detected. Polymorphism is present in the haptoglobin, transferrin and protease inhibitor systems. Two variant alleles, TfDchi and TfB are present in the transferrin system, but the B variant has not been identified. Similarly, 3 persons with caeruloplasmin variants were found, but also these variants have not been identified. No abnormal haemoglobins were detected. All other systems revealed the presence of only normal phenotypes.