Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli

Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed. From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained. Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1. It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3. This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells. Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene. Thus, the purified h-TR beta 1 is active. To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized [3',5'-125I]T4. A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing. Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.     

Preventive effect of angiotensin I on weight reduction in the adrenal glands of DOCA/salt hypertensive rats

We examined the effect of angiotensin I (AI), without the effect of angiotensin II (AII) converted from AI, on the weight of the adrenal glands, adrenal corticosterone (B) and adrenal aldosterone under conditions where the renin-angiotensin system was suppressed, since a reduction in the size of the adrenal glands is often observed in DOCA/salt hypertensive rats. Sixty male Wistar rats fed on a 1% NaCl solution were divided into 6 groups as follows: a) Salt group: received sesame oil and vehicle, b) Salt + C group: received sesame oil and MK422 (0.14 mg/day), an angiotensin converting enzyme inhibitor (CEI), c) DOCA group: received DOCA (30 mg/week) and vehicle, d) DOCA + A group: received DOCA and AI (0.5 mg/kg/day), e) DOCA + A + C group: received DOCA and AI with MK422, and f) DOCA + C group: received DOCA and MK422. After 4 weeks, the rats were sacrificed to sample their blood and remove their adrenal glands. There was no significant difference in adrenal B among the groups apart from the DOCA + C group. Adrenal aldosterone was lower in the groups of DOCA/salt hypertensive rats than in the Salt group and Salt + C group. Furthermore, the DOCA + A + C group and DOCA + C group had lower adrenal aldosterone levels than the DOCA group and DOCA + A group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlling mechanism of ATP regeneration by glycolytic pathway in a yeast: Hansenula jadinii

Summary The regulatory mechanism of ATP regeneration by the glycolytic pathway in Hansenula jadinii cells was investigated by analyzing the initial stage of CDP-choline fermentation. As a result, the on-off of ATP regeneration was found to be determined by the ATP concentration overcoming the inhibitory effect of phosphate buffer on hexokinase activity. The concentration of ATP at the initial stage of fermentation was greatly influenced by the kinds and amounts of glycogen in cells. Based on these results, the regulatory mechanism of ATP regeneration by the glycolytic pathway is discussed in detail.     

Age-dependent modifications of lipogenic enzymes

When 1-, 2- and 9-month-old rats previously adapted to a commercial stock diet were fed a fat-free diet (induction) and also when the process was reversed (repression), the turnovers of lipogenic enzymes in liver were measured by following time courses for the change of the enzyme activities. The magnitudes of the induction of glucose-6-phosphate dehydrogenase, malic enzyme and acetyl-CoA carboxylase were very high in 1-month-old rats and then decreased with aging. In the induction phase, the rates of synthesis of the enzymes were markedly decreased with increasing age as compared with the rate constants of degradation. The age-dependent alterations were not shown so much in the repression phase as in the induction phase. It is suggested that the age-dependent impairment in induction may be due to some alterations in the enzyme-forming system.     

Maturation process of secondary ossification centers in the rat and assessment of bone age.

The maturation process from the appearance to the fusion of the secondary ossification centers of extremities was studied in Wistar rats aged 0 to 134 weeks. The examination of the secondary ossification centers made by radiography. The assessment of the stage of development was made in accordance with the criteria proposed by Ohwada and Sutow. The secondary ossification center was found to be take one of the following three types of maturation processes : (1) the acute ossification, (2) the delayed ossification, and (3) the incomplete ossification. No fusion was observed up to 134 weeks in certain epiphyses of the rat. This type of ossification designated as the incomplete ossification may be specific to the mouse and rat. The relative lengths of time required for appearance and fusion in the average prospective life were obtained for the rat. They were compared with those of the mouse and man. The relative length of time necessary for maturity of the secondary ossification centers was shown to be the shortest in the rat and the longest in man. The results suggested that the rat may reach maturity in the bone age at 17 to 21 weeks of age. The rat at this age may be regarded as being adult corresponding to age 17 weeks in mice and 18 to 24 years in man.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Summary Feulgen nuclear staining with pararosanilin-SO₂ was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell.The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality beween the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37° C for 10 h.The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure.Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.Partly supported by Deutsche Forschungsgemeinschaft (DFG), grant Nr. Bo 395/4     

Process of infection with bacteriophage phi chi 174. XL. Viral DNA replication of phi chi 174 mutants blocked in progeny single-stranded DNA synthesis.

Mutation in several different cistrons of bacteriophage phi chi 174 blocks net progeny single-stranded DNA synthesis at the late period of infection (15). For the study of the functions of these cistrons in single-stranded DNA synthesis, asymmetric replication of replicative form DNA was examined at the late period of infection with amber mutants of these cistrons. While the normal, rapid process of asymmetric single-stranded viral DNA synthesis is blocked at the late period of these mutant infections, an asymmetric synthesis of the viral strand of replicative-form DNA is observed in this period, though at a reduced level, together with degradation of prelabeled viral strand. Some intermediate replicative-form molecules were also detected. Asymmetric synthesis of the viral strand of replicative-form DNA at the late period of phi chi infection is completely inhibited in the presence of a low concentration (35mug/ml) of chloramphenicol (which also blocks net single-stranded viral DNA synthesis). These results are discussed in terms of the possible role of the specific viral proteins for normal single-stranded DNA synthesis.