Expression of tuna growth hormone cDNA in Escherichia coli

Summary In order to produce tuna (Thunnus thynnus) growth hormone (GH), expression plasmid (pUES13S) carrying tuna GH cDNA was constructed using a vector (pKK223-3), in which the replication origin was replaced with that of pUC19. The expression of the tuna GH cDNA was greatly affected by the distance between a Shine-Dalgarno (SD) sequence and the initiation codon (ATG) and was most efficient when the distance was adjusted to 13 base pairs (bp). The amount of tuna GH produced by Escherichia coli JM109 with pUES13S was more than 12.5% of the total cytosolic proteins and the product was immunologically identified to be tuna GH (mol. wt. 21 000) by Western blot analysis using tuna GH specific immunoglobulin G (IgG). Another plasmid (pUES13S-2) containing tandemly polymerized tuna GH cDNA was constructed, to improve the productivity of tuna GH. When E. coli JM109 carrying pUES13S-2 was incubated at 40°C, the amount of tuna GH produced reached about 20% of the total cytosolic proteins.     

High affinity Zn2+ inhibitory site(s) for the trypsin-like peptidase of the 20S proteasome

The effect of Zn²⁺ on three major peptidase activities of the 20S proteasome purified from Xenopus oocytes was kinetically investigated. An extremely low concentration of Zn²⁺ (μM range) strongly inhibited the trypsin-like activity of the 20S proteasome which was fully recoverable by the addition of EDTA. The concentration of Zn²⁺ for half-maximum inhibition (K_0.5) was 0.60 μM which was at least 10 times lower than that of any other divalent cation tested and essentially the same as for proteasomes purified from various other organisms indicating that the inhibition is highly Zn²⁺-specific, reversible, and common to the proteasome regardless of its source. Zn²⁺ at concentrations below 100 μM instantaneously activated the chymotrypsin-like and PGPH activities, and the Zn²⁺ concentration for half-maximum activation was found to be 42-48 μM. These activities were time-dependently inactivated by submillimolar concentrations of Zn²⁺. The inactivation rates were dependent on the concentration of Zn²⁺ and reached a maximum of 1.60-2.40 min⁻¹ for the three peptidase activities under the conditions used. The Zn²⁺ concentration for half-maximum inactivation was found to be 0.70-1.23 mM. This time-dependent inactivation was not reversed by the addition of EDTA or DTT and might not be accompanied by the dissociation of subunits of the 20S proteasome indicating that all activities are inactivated by an identical phenomenon. These results reveal the three types of effects of Zn²⁺ on the 20S proteasome.     

The nuclear export signal (NES) found in the amino-terminal region of carp MEK1 and MKK6 is lacking in carp MKK4

Carp MKK4 (cMKK4) cDNA was isolated from an ovary cDNA library. cMKK4 mRNA was ubiquitously distributed in various tissues of adult carp. Sequence analysis revealed that cMKK4 lacks a nuclear export signal sequence, unlike mammalian and frog MEKs (ERK activator) and cMKK6 (carp p38 activator), where it plays an important role in anchoring these MAP kinase activators to the cytoplasm. cMKK4 protein was found to be diffused throughout the cell, whereas cMEK1 and cMKK6 proteins were seen exclusively in the cytoplasm.     

Cross-sectional geometric properties along the diaphysis of femur and humerus in chimpanzees and humans

The cross-sectional geometric parameters were determined serially along the diaphysis of 3 paired humeri and femora of chimpanzees by using the computed X-ray tomographic scans, and compared with those of humans. In magnitude, the femoral parameters were greater and humeral parameters were less, respectively, in humans than in chimpanzees. While the changing pattern among the parameters along the diaphysis was very similar both in the femur and humerus of chimpanzees, the pattern in the humans was reversed between the cross-sectional area and area moments of inertia. In chimpanzees, the femoral parameters increased toward the most proximal diaphysis, whereas humeral parameters yielded a moderate peak in a portion slightly proximal to mid-shaft. Potential mechanisms responsible for these findings were discussed.     

Effects of fixation and preservation conditions on immunohistochemical profiles of the skeletal muscle fibers in Japanese macaques

Effects of fixation and preservation conditions of muscle tissues on immunohistochemical profiles are investigated. Samples of the hind limb and epaxial muscles were removed from 4 adult female Japanese macaques (Macaca fuscata) fixated with 10% formalin and preserved in the same solution under different conditions for 6 months to 4 years and 6 months. Sections were stained with indirect immunofluorescence and avidin-biotin peroxidase complex methods using an antibody against fast myosin (Mouse Monoclonal Anti-skeletal Myosin-Fast, clone MY-32, Sigma) as a primary antibody. Clear responses to the antibody were demonstrated in the samples from the specimens fixated by injection or immersion with 10% formalin and preserved in the same solution for 6 months to 1 year and 6 months. Distribution patterns of the fibers reacting to the antibody coincided with that of the fast twitch fibers determined using enzyme-histochemical techniques in these samples. Clear responses to the antibody were not demonstrated in the samples from the specimen repeatedly rinsed in water for gross anatomical dissections during the preservation period. The results of this study warrant applications of immunohistochemical techniques to the study of fiber type composition in muscle samples from specimens fixated with formalin and preserved in the same solution for a long term.     

Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.     

Differential regulation of diacylglycerol kinase isozymes in cardiac hypertrophy

To examine the involvement of diacylglycerol kinase (DGK) and phosphatidic acid phosphatase (PAP) in pressure overloaded cardiac hypertrophy, rats were subjected to either ascending aortic banding for 3, 7, and 28 days or sham operation. In comparison with sham-operated rats, the left ventricular (LV) weight of the aortic-banded rats increased progressively. At 28 days after surgery, the expression of DGKepsilon mRNA but not DGKzeta or PAP2b mRNA in the LV myocardium significantly decreased in the aortic-banded rats compared with the sham-operated rats. DGKzeta protein in the LV myocardium translocated from the particulate to the cytosolic compartment in the aortic-banded rats. Furthermore, the myocardial content of 1,2-diacylglycerol and PKCdelta protein expression in the particulate fraction of the LV myocardium significantly increased in aortic-banded rats compared with sham-operated rats. These results suggest that DGKepsilon and DGKzeta play distinct roles in the development of pressure overloaded cardiac hypertrophy and that the two isozymes are differentially regulated.     

Association of paracellin-1 with ZO-1 augments the reabsorption of divalent cations in renal epithelial cells

Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether (45)Ca(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (Delta TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the Delta TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of (45)Ca(2+) transport from apical to basal compartments, compared with those expressing the Delta TRV, ARV, and TRA mutants and the mock cells. (45)Ca(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced (45)Ca(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1.ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.     

Reconstitution of Sperm Nuclei of Zebrafish (Danio rerio) in Xenopus Egg Extracts

Cell-free extracts of Xenopus eggs cause cyclic change in permeabilized sperm nucleus, nuclear envelope breakdown, chromosome condensation, and reformation of nuclei. In this study, the ability of cell-free extracts to cause similar changes in zebrafish sperm was examined. When lysolecithin-treated sperm from zebrafish were incubated in Xenopus egg extracts, a series of changes in sperm nuclear morphology were observed periodically. These changes correlated with maturation-promoting factor (MPF) activity. Furthermore, sperm nuclei of zebrafish replicated DNA during reconstitution in Xenopus egg extracts. These results showed that cell-free extracts of Xenopus egg possess the ability to cause cell-cycle-dependent changes in zebrafish sperm, implying the possibility of generating transgenic zebrafish in a similar way to transgenic Xenopus. Received October 21, 1999; accepted July 18, 2000.