Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase.

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 x 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5'-[gamma-thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.     

Interesterification selectivity in lipase catalyzed reactions of low molecular weight triglycerides

Summary In the absence of an organic solvent, a buffer to enzyme weight ratio of 1 gives maximum selectivity to interesterification over hydrolysis in a lipase-catalyzed mixture of triacetin and tributyrin. Addition of hexane enhances interesterification as does a reversed micelle configuration.     

Estimation of oxygen penetration depth in immobilized cells

Summary A simple method is proposed for calculating oxygen pentration depth in immobilized cells by assuming zero order kinetics in the presence of several external oxygen transport resistances. Calculations indicate that typical penetration depths of oxygen for immobilized microbial cells are in the range of 50–200 and those for immobilized or encapsulated animal and plant tissue culture are about 500–1000 . Based on calculations, oxygen transport in microencapsulation and microcarriers for tissue cultures are not transport-limited, but a slight limitation is expected for those in a hollow fiber reactor.Nomenclature a_s specific area of a support (cm) - Bi Biot number - dimensionless - C_b oxygen concentration in the bulk liquid (mM) - C _b C _b ^* -C_cr (mM) - C _b ^* bulk oxygen concentration in equilibrium with air (mM) - C_cr critical oxygen concentration (mM) - C_s oxygen concentration in the solid phase (mM) - d_p diameter or thickness of a support (cm) - D_eff effective diffusivity of oxygen in the solid phase (cm²/s) - k_m membrane permeability of oxygen (cm/s) - k _m ^* D_eff/_m - k_La_L liquid phase mass transfer rate coefficient (1/s) - k_sa_s solid phase mass transfer rate coefficient (1/s) - (OUR)_v volumetric oxygen uptake rate (mmol O₂/l) - p geometry parameter, p=0 for slab, p=1 for cylinder, p=2 for sphere - P_d oxygen penetration depth (cm) - P _d oxygen penetration depth in the absence of external diffusion limitation (cm) - Q volumetric oxygen uptake rate, (mmol O₂/l·h) - specific oxygen uptake rate (mmol O₂gm biomass (dry)·h) - r length coordinate (cm) - r_c oxygen penetration depth for sphere (cm) - r _c r_c in the absence of external diffusion limitation (cm) - r _c ^* oxygen penetration depth for cylinder (cm) - r _c ^* r _c ^* in the absence of external diffusion limitation (cm) - r_com combined mass transfer rate resistance (s) - r_d location where C_s becomes zero or C_cr (cm) - r_i radius of cylinder or sphere, half thickness of slab (cm) - U_sg superficial gas velocity (cm/s) - X cell concentration (g/l) Greek letters Thiele modulus, dimensionless - _L, _s liquid and solid phase volume fraction, respectively, dimensionless - effectiveness factor On sabbatical leave from KAIST, Seoul, Korea     

Different patterns of Epstein-Barr virus gene expression and of cytotoxic T-cell recognition in B-cell lines infected with transforming (B95.8) or nontransforming (P3HR1) virus strains

Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines have been converted to EBV genome positivity by in vitro infection with the transforming EBV strain B95.8 and with the nontransforming mutant strain P3HR1, which has a deletion in the gene encoding the nuclear antigen EBNA2. These B95.8- and P3HR1-converted lines have been compared for their patterns of expression of EBV latent genes (i.e., those viral genes constitutively expressed in all EBV-transformed lines of normal B-cell origin) and for their recognition by EBV-specific cytotoxic T lymphocytes (CTLs), in an effort to identify which latent gene products provide target antigens for the T-cell response. B95.8-converted lines on several different EBV-negative BL-cell backgrounds all showed detectable expression of the nuclear antigens EBNA1, EBNA2, and EBNA3 and of the latent membrane protein (LMP); such converts were also clearly recognized by EBV-specific CTL preparations with restriction through selected human leukocyte antigen (HLA) class I antigens on the target cell surface. The corresponding P3HR1-converted lines (lacking an EBNA2 gene) expressed EBNA1 and EBNA3 but, surprisingly, showed no detectable LMP; furthermore, these converts were not recognized by EBV-specific CTLs. Such differences in T-cell recognition were not due to any differences in expression of the relevant HLA-restricting determinants between the two types of convert, as shown by binding of specific monoclonal antibodies and by the susceptibility of both B95.8 and P3HR1 converts to allospecific CTLs directed against these same HLA molecules. The results suggest that in the normal infectious cycle, EBNA2 may be required for subsequent expression of LMP and that both EBNA2 and LMP (but not EBNA1 or EBNA3) may provide target antigens for the EBV-specific T-cell response.     

Serum cortisol radioimmunoassay values in the normal ferret and response to ACTH stimulation and dexamethasone suppression tests

Cortisol values were obtained from 39 ferrets, Mustela putorius furo, by using a commercial radioimmunoassay. Sera from 25 males (18 intact, 7 neutered) and 14 females (7 intact, 7 spayed) were assayed. Resting serum cortisol values ranged from 0.13 to 2.70 micrograms/dl for males (mean = 0.93 +/- 0.63 micrograms/dl), and 0.55 to 1.84 micrograms/dl for females (mean = 0.86 +/- 0.29 microgram/dl). The resting cortisol values of both males and females were comparable to those of the cat (1.0 to 3.0 micrograms/dl). A 7 year old male ferret with suspected hyperadrenocorticism and an adrenal mass had a cortisol level of 8.1 micrograms/dl. Adrenal cortical carcinoma was the histologic diagnosis. One adult female ferret had a cortisol level of 3.30 micrograms/dl. This animal also had proliferative colitis upon necropsy. An ACTH stimulation test (1 U/kg IM) and a low-dose dexamethasone suppression test (0.1 mg/kg) were performed on 10 ferrets. Post-ACTH serum cortisol levels increased by an average of 89%. Post-dexamethasone serum cortisol values decreased by an average of 18% 6 hours post-injection.     

Transformations and fate of sheep urine-N applied to an upland U.K. pasture at different times during the growing season

The fate of sheep urine-N applied to an upland grass sward at four dates representing widely differing environmental conditions, was followed in soil (0–20 cm) and in herbage. Urine was poured onto 1-m² plots to simulate a single urination in August 1984 (warm and dry), May (cool), July and August 1985 (cool and wet) at rates equivalent to 40–52 g N m⁻². The transformation of urine-N (61–69% urea-N) in soil over a 6–7 week period followed the same general pattern when applied at different times during the season; rapid hydrolysis of urea, the appearance of large amounts of urine-N as ammonium in soil extracts, and the appearance of nitrate about 14 days after application. The magnitude of “apparent” nitrification however differed markedly with environmental conditions, being greatest in May 1985 when a maximum of 76% of the inorganic soil N was in the form of nitrate. At all other application dates nitrate levels were relatively low. With the August 1984 application soil inorganic N returned to control levels (given water only) after 31 days but considerable amounts remained in soil for 60–90 days with the other applications. Weekly cuts to 3-cm indicated that increases in herbage dry matter and N yields in response to urine application were greatest in absolute terms after the May 1985 application and continued for at least 70 days with all applications. Relative to control plots the May application resulted in a 3-fold increase in herbage DM compared with corresponding values of 6-, 5-, and 7-fold increases with the August 1984, July and August 1985 applications. Recovery of urine-N in herbage was poor averaging only 17% of that applied at different dates, while recovery in soil extracts was incomplete. The exact routes of loss (volatilisation, leaching, denitrification or immobilisation) were not quantified but it is evident that substantial amounts of urine-N can be lost from the soil-plant system under upland conditions.     

Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors

Two distinct v-erbA-related cDNA clones representing the products of different genes were isolated from a rat liver cDNA library. The first, rc-erbA-alpha, was 82% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 45,000 daltons. This cDNA clone arises from the same gene product as a v-erbA-related cDNA isolated from rat brain by Thompson et al. (Thompson, C. C., Weinberger, C., Lebo, R., and Evans, R. (1987) Science 237, 1610-1614). The second cDNA clone, rc-erbA-beta, was 76% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 52,000 daltons. Both rc-erbA-alpha and rc-erbA-beta translational products bound 3,5,3'-triiodo-L-thyronine with affinities equal to each other (Kd approximately equal to 0.4 nM) and comparable to the nuclear thyroid hormone receptor extracted from rat liver. The relative affinities of a series of thyroid hormone analogs for both translational products were also identical. In various tissues and cell lines, the relative levels of rc-erbA-beta RNA, but not rc-erbA-alpha RNA, correlated with measurements of nuclear 3,5,3'-triiodo-L-thyronine binding sites. Based on this correlation, we suggest that rc-erbA-beta may encode the "classical" nuclear thyroid hormone receptor, whereas rc-erbA-alpha may encode an isoreceptor species with differing functional properties.     

Sequence specificity of 125I-labelled Hoechst 33258 in intact human cells

Using polyacrylamide/urea DNA sequencing gels, the DNA sequence selectivity of 125I-labelled Hoechst 33258 damage has been determined in intact human cells to the exact base-pair. This was accomplished using a novel procedure with human alpha RI-DNA as the target DNA sequence. In this procedure, after size fractionation, the alpha RI-DNA is selectively purified by hybridization to a single-stranded M13 clone containing an alpha RI-DNA insert. The sequence specificity of [125I]Hoechst 33258 was indistinguishable in intact cells from purified high molecular weight DNA; and this is surprising considering the more complex environment of DNA in the nucleus where DNA is bound to nucleosomes and other DNA binding proteins. The ligand preferentially binds to DNA sequences which have four or more consecutive A.T base-pairs. The extent of damage was measured with a densitometer and, relative to the damage hotspot at base-pair 94, the extent of damage was similar in both purified high molecular weight DNA and intact cells. [125I]Hoechst 33258 causes only double-strand breaks, since single-strand breaks or base damage were not detected. These experiments represent the first occasion that the sequence specificity of a DNA damaging agent, which causes only double-strand breaks, has been determined to the exact base-pair in intact cells.