Cloning of cDNAs encoding sorbitol dehydrogenase-2a and b, enzymatic characterization, and up-regulated expression of the genes in Bombyx mori diapause eggs exposed to 5 °C

We previously cloned a cDNA for sorbitol dehydrogenase (SDH1) from Bombyx mori. In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and SDH2b). The amino acid sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to B. mori SDH1. The SDH2ab and SDH1genes were located in tandem within about 40 kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5 °C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25 °C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the SDH activity seen in diapause eggs exposed to 5 °C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in Escherichia coli and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4∼9.0, and the Km value for sorbitol was 12.6 mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified SDH from B. mori diapause-terminated eggs exposed to 5 °C for 300 days which showed higher SDH activity using two-step affinity chromatography. The highly purified SDH showed a higher Km value (125 mM) for sorbitol, being similar to the value (136 mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no SDH genes other than SDH1 and 2ab are present in B. mori genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased SDH activity seen in diapause eggs after acclimation to 5 °C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original SDH gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and SDH2b genes.     

Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C

To understand the molecular mechanisms of how 5 °C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5′-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (−95 to −58) and -2 (−145 to −121), and SamuiHSE-1 (−84 to −55) and -2 (−304 to −290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5 °C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6–8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5 °C.     

Phosphorylation of glycogen synthase kinase-3β in relation to diapause processing in the silkworm, Bombyx mori

Glycogen synthase kinase-3 (GSK-3) is a multifunctional protein kinase that plays important roles in regulating both glycogen synthesis and protein synthesis. In the present study, we investigated GSK-3β phosphorylation of silkworm eggs by immunoblotting with a conserved phospho-specific antibody to GSK-3β. Results showed that the temporal changes in GSK-3β phosphorylation were closely related to changes in glycogen levels previously reported by other researchers. In diapause eggs, an abrupt decrease in phosphorylation of GSK-3β was found with the onset of diapause, and phosphorylation level of GSK-3β reached a minimum level within 1 week after oviposition. However, when diapause eggs were incubated at 25 °C for 15 days and then transferred to 5 °C, a great increase in GSK-3β phosphorylation was observed 5 days after transfer to 5 °C and high levels were maintained throughout the chilling period. In both non-diapause eggs and eggs whose diapause initiation was prevented by HCl, levels of GSK-3β phosphorylation appeared to remain relatively high for several days and then greatly decreased 2 or 3 days before hatching. Moreover, GSK-3β phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, did not inhibit GSK-3β phosphorylation in dechorionated eggs, although U0126 dose-dependently inhibited ERK phosphorylation. This result showed that ERK phosphorylation is not involved in upstream signaling for GSK-3β phosphorylation and that there may be two distinct signaling pathways involved in diapause processing in Bombyx mori eggs.     

Characterization of the guanosine 3′∶5′-monophosphate-dependent protein kinase from silkworm eggs and analysis of the endogenous protein substrates

Summary In a previous paper, two types of cyclic nucleotide-dependent protein kinases, namely cGMP dependent G-kinase and cAMP dependent A-kinase, in silkworm eggs has been reported (Takahashi et al. 1975; Takahashi 1976). One of these, G-kinase, has now been purified 2400-fold by means of ammonium sulfate fractionation, chromatography on hydroxylapatite, DEAE cellulose, and gel filtration.Some of the properties of the enzyme are described. The enzyme is highly dependent on cGMP; it is strongly inhibited by GTP in a noncompetitive manner not only for ATP but also for cGMP. GTP was found to be highly inhibitory on G-kinases from various tissues of the silkworm, but did not inhibit the A-kinase.Incubation of the egg extract with [-³²P]ATP and Mg²⁺ led to the formation of three major³²P-labelled proteins, with molecular weights of 42.000, 70.000 and 180.000 as analyzed by SDS polyacrylamide gel electrophoresis. Two of them corresponded to the subunits of vitellin.The silkworm vitellin was effectively phosphorylated both by the highly purified G-kinase and by the A-kinase. It is concluded that the G-kinase is involved in the phosphorylation of vitellin in developing silkworm eggs.Abbreviations cAMP adenosine 35-monophosphate - cGMP guanosine 35-monophosphate - A-kinase adenosine 35-monophosphate-dependent protein kinase - G-kinase guanosine 35-monophosphate-dependent protein kinase - MIX 1-methyl-3-isobutylxanthine     

Catalytic activity and acyl-chain selectivity of diacylglycerol kinase ɛ are modulated by residues in and near the lipoxygenase-like motif

Diacylglycerol kinase (DGK) ? plays an important role in the resynthesis of phosphatidylinositol by mediating the phosphorylation of diacylglycerol to phosphatidic acid. DGK? is unique among mammalian DGK isoforms in that it is the only one that shows acyl-chain selectivity, preferring diacylglycerols with an sn-2 arachidonoyl group. The region responsible for this arachidonoyl specificity is the lipoxygenase (LOX)-like motif found in the accessory domain, adjacent to DGK?'s catalytic site. Many mutations within the LOX-like motif result in a loss of enzyme activity. However, the few mutants that retain significant activity exhibit some decrease in selectivity for the arachidonoyl chain. In the present work, we have explored mutations in a region adjacent to the LOX-like motif, which is also contained within the same hydrophobic segment of the protein. This adjacent region also contains a cholesterol recognition/interaction amino acid consensus motif. Being outside of the LOX-like motif, this region likely has less direct contact with the substrate, and more activity is retained with mutations. This has allowed us to probe in more detail the relationship between this region of the protein and substrate specificity. We demonstrate that this cholesterol recognition/interaction amino acid consensus domain also plays a role in acyl-chain selectivity. Despite the high degree of conservation of the amino acid sequence in this region of the protein, certain mutations result in proteins with higher activity than the wild-type protein. These mutations also result in a selective gain of acyl-chain preferences for diacylglycerols with different acyl-chain profiles. In addition to the LOX-like motif, adjacent residues also contribute to selectivity for diacylglycerols with specific acyl-chain compositions, such as those found in the phosphatidylinositol cycle.     

Guanosine 3′,5′-monophosphate-dependent protein kinase from the silkmoth,Bombyx mori: Further evidence for the involvement of kinase in the phosphorylation of vitellin and the effect of phosphorylation on the susceptibility of vitellin to a cysteine proteinase in the egg

• 1.1. In the developing silkmoth eggs, a high level of activity of a cGMP-dependent protein kinase (G-kinase) is found. Vitellin (Vn) is an excellent substrate for the kinase, suggestin that the kinase may be involved in the phosphorylaiton of Vn in intact developing eggs.
• 2.2. To characterize this system in greater detail, the following experiments were performed: (a) changes in the levels of cyclic nucleotides and changes in cyclic nucleotide-dependent protein kinase activities were monitored in the eggs; (b) ³²P-o-phosphate was microinjected into eggs to demonstrate the phosphorylation of Vn in vivo; (c) to determine the role, if any, in vivo of the phosphorylation of Vn, the effect of phosphorylation on the susceptibility of Vn to a cysteine proteinase after the phosphorylation of Vn by G-kinase was studied.
• 3.3. The results revealed that the phosphorylation of Vn serves as the trigger for the proteolytic digestion of Vn during the developments, and may be able to provide the signal for the degradtion of Vn by the cysteine proteinase.

     

Permeability of the human erythrocyte to glycerol in 1 and 2 m solutions at 0 or 20 °C

There are increasing numbers of exceptions to a central tenet in cryobiology that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to determine the amount of solute that has permeated a cell prior to freezing. The amount in human red cells was estimated from the flux equation $\text{ds}\text{dt}\text{= P}{}_{\mathrm{<mtext>\gamma </mtext>}}\text{A}$[(activity external solute) — (activity internal solute)]. Solving the equation required knowledge of P_γ the permeability constant for the solute. Estimates of P_γ for glycerol were made in two ways: (i) by measuring the time to 50% hemolysis of human red cells suspended in 1 or 2 m solutions of glycerol that were hypotonic with respect to NaCl, and (ii) by measuring the time required for red cells in 1 or 2 m solutions of glycerol in isotonic saline-buffer to undergo osmotic shock upon tenfold dilution with isotonic saline-buffer. The measurements were made at 0 and 20 °C. The values of P_γ were about 2.5 × 10^?4 cm/min at 20 °C and about 0.9 × 10^?4 cm/min at 0 °C. The difference corresponds to an activation energy of 7.2 kcal/mole. These values of P_γ are 100 to 600 times higher than those for glycerol permeation in the bovine erythrocyte. The values of P were relatively unaffected by whether calculations were based on classical or irreversible thermodynamics and by the choice of concentration units in the flux equations. Calculations of the kinetics of glycerol entry using these P values showed that the concentration of intracellular glycerol reaches 90% of equilibrium in 1.2 min at 0 °C and in 0.6 min at 20 °C. The osmolal ratio of intracellular glycerol to intracellular nonpermeating solutes reaches 90% of equilibrium in 7 min at 0 °C and in 3.2 min at 20 °C.     

Protein kinase Cβ isoform down-regulates the expression of MDR3 P-glycoprotein in human Chang liver cells

The MDR3 protein is a transporter of phosphatidylcholine on the canalicular membrane of human hepatocytes. Previously we showed that the expression of MDR3 mRNA was down-regulated by phorbol 12-myristate 13-acetate (PMA) in human Chang liver cells. In the present study, to elucidate the isoform of protein kinase C (PKC), which influences the level of MDR3 protein, we investigated the effects of PKC-specific inhibitors and antisense oligonucleotides. The level of protein decreased around 50% after treatment for 3–5 days using the dosage of PMA effective against the mRNA expression. The half-life of the MDR3 protein was estimated to be about 5 days. This decrease was antagonized by GF109203X, a non-selective inhibitor of PKCs, and Gö6976, a selective inhibitor for PKCα/β. These inhibitors also suppressed the reduction in MDR3 protein. To specify the isoform of PKC, the cells were treated with antisense oligonucleotide of PKCα or PKCβ. The suppressive effects on MDR3 mRNA of PMA were attenuated in antisense PKCβ-treated cells, but those in antisense PKCα-treated cells were not attenuated. These suggested that PKCβ plays a regulatory role in the expression of MDR3.     

Simultaneous decrease in ammonia and hydrogen sulfide inhibition during the thermophilic anaerobic digestion of protein-rich stillage by biogas recirculation and air supply at 60 °C

We had proposed a novel method to reduce ammonia inhibition during thermophilic anaerobic digestion via recirculation of water-washed biogas into the headspace (R1 system) or liquid phase (R2 system) of reactors. The feasibility of reducing the ratio of recirculated biogas to biogas produced (called the biogas recirculation ratio) was investigated in the present study. Thermophilic anaerobic digestion at 53 °C and 60 °C with a biogas recirculation ratio of 150 facilitated stable digestion performance and biogas production at a higher organic loading rate of 7 g/L/d in the R1 system, while the ammonia removal efficiency increased 1.23-fold when the temperature increased from 53 °C to 60 °C. At 60 °C, the biogas recirculation ratios in the R1 and R2 systems decreased to 50 and 10, and the ammonia absorption rates were 6.1 and 8.3 mmol/L/d, respectively, without decreasing the anaerobic digestion performance. The ammonia absorption rate of 8.3 mmol/L/d in the R2 system was higher than the rate of 7.8 mmol/L/d at the biogas recirculation ratio of 150 in the R1 system. The hydrogen sulfide content in the biogas was reduced to less than 50 ppm by supplying air at 3% of the amount of biogas produced into the reactor.     

A comparison of the drying of chymotrypsin bound to bead cellulose by lyophilisation and in air at 25 to 70°C

Summary The effect was studied of the method of drying chymotrypsin attached to bead cellulose on its chemical and physical characteristics. These characteristics are not deteriorated when replacing the commonly used lyophilisation by fluid drying in the air stream. The preparations saved essentially the same proteolytic activity as well as stability even when increasing the drying air temperature to 70°C.     

Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula

Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3β4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the β4 subunit is rate limiting for receptor activity, and that current increase by β4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a β4-specific residue (S435), mapping to the intracellular vestibule of the α3β4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the?medial habenula (MHb). Thus, this study both provides insights into α3β4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes.     

Glutamine synthetase in cultured whole retinas from the embryonic chick: Enhancement of basal and induced activity by 4 °C storage

Storage of whole retinas from the embryonic chick for 24 h at 4 °C resulted in increased basal levels of glutamine synthetase (GS) during subsequent incubation at 37 °C in the absence of cortisol. GS levels in these retinas maintained initially at 4 °C (CS), in many cases, exceeded GS levels in cortisol-induced whole retinas incubated solely at 37 °C. The increase in basal GS activity is seen within 48 h of the transfer of the retinas from 4 to 37 °C. If cortisol (0.001 μg/ml = 2.8 nm or 0.01 μg/ml = 28 nm) is added during the last 24 h of culture to CS retinas subsequently transferred to 37 °C, levels of GS are attained that are higher than those in the corresponding retinas cultured continually at 37 °C. However, the activity ratios (GS specific activity in cortisol-treated retinas/GS specific activity in retinas not exposed to cortisol) are similar for CS retinas and those maintained at 37 °C throughout. Monolayers of retinal cells display similar basal and cortisol-induced levels of GS independent of treatment. Retinal monolayers maintained at 4 °C for 24 h and subsequently incubated at 37 °C do not exhibit increases in either basal or cortisol-induced levels of GS over those in monolayers maintained at 37 °C throughout. The CS-promoted increase in the basal and cortisol-induced GS activity of whole retinas is eliminated by enzymatic dispersion of the retina just prior to 37 °C culture of the cells as monolayers. Both basal and cortisol-induced GS levels in the latter monolayers resemble those in retinal cells kept as monolayers throughout.     

Protein kinase activity stimulated by adenosine 3′:5′-cyclic monophosphate in synaptic-membrane fragments from ox brain. Inhibition of intrinsic activity by free and membrane-bound calcium ions

1. Cyclic AMP-stimulated protein kinase activity phosphorylating intrinsic substrates in preparations of synaptic-membrane fragments from ox cerebral cortex was examined in relation to (a) the content of membrane-bound Ca(2+) in the preparations and (b) added Ca(2+) in the assay medium. 2. Centrifugal washing of synaptic-membrane fragments with buffered ethane dioxybis(ethylamine)tetra-acetate solutions decreased bound Ca(2+) from 2.8+/-0.4 (s.d.) to 0.9+/-0.3nmol/mg of protein. In washed preparations basal protein kinase activity was increased by about 40% and the cyclic AMP-stimulated activity by about 15%. Addition of Ca(2+) in the concentration range 5-50mum to the assay medium progressively inhibited the kinase activity of the washed preparations; in this range of Ca(2+) concentration the basal activity was inhibited more than the stimulated activity. 3. In unwashed preparations concentrations of Ca(2+) above 100mum inhibited the cyclic AMP-stimulated activity more than the basal activity. 4. The inhibitory effect of several concentrations of Ca(2+) was examined in relation to cyclic AMP concentration; no evidence for competition between Ca(2+) and cyclic AMP for a site on the enzyme was observed.     

Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the “in cooling” period (5 °C)

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100 × 10⁶ sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (−20 °C/min to −100 °C and stored at −196 °C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters – by CASA –, and viability (VIAB), viable and non-apoptotic status (YOPRO−), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) – by flow cytometry –. In Experiment 1, we assessed different storage times (0, 0.5, 1 – control –, 4–5, 7–8 and 11–12 h) at 5 °C from final dilution to freezing. After thawing, non-equilibrated samples (0 h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72 h) at 5 °C before freezing using storage for 1 h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48–72 h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24 h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.     

Superoxide dismutase (SOD) in boar spermatozoa: Purification,biochemical properties and changes in activity during semen storage (16 °C) in different extenders

The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16 °C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16 °C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67 kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45 °C. The enzymatic activity of form released after cold shock was inhibited by H₂O₂ and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H₂O₂ and DDC (40%). The molecular form released after urea treatment was a 30 kDa protein with maximum activity at 20 °C and pH 10. Enzymatic activity of this form was inhibited by H₂O₂ by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant.