Mouse phosphoinositide 3-kinase p110alpha gene: cloning, structural organization, and localization to chromosome 3 band B.

Phosphoinositide 3-Kinases (PI3-Kinases) are a family of dual specificity enzymes with a unique lipid kinase activity toward the D-3 position of the inositol ring of phosphoinositides and a less well characterized serine/threonine protein kinase activity. Class IA PI3-Kinases comprise a 110-120 kDa catalytic subunit (usually termed p110) and an 85 kDa or 50 to 55 kDa regulatory subunit (often called p85). cDNAs for three mammalian Class IA PI3-Kinase catalytic subunits designated p110alpha, p110beta, and p110delta have been cloned from several species. A YAC clone for the human p110alpha gene has also been cloned and mapped to chromosome 3q26.3. However, structural organization for any of the PI3-Kinase p110alpha genes has not been reported. Here, we report the cloning, structural organization, and chromosomal localization of the mouse PI3-Kinase p110alpha gene. The translated portion of the mouse p110alpha gene is encoded by 19 exons that span at least 24 kb. Dual color fluorescence in situ hybridization (FISH) was performed to determine the chromosomal localization of the mouse PI3-Kinase p110alpha gene. FISH results and DAPI banding demonstrated localization of the p110alpha gene to band B on mouse chromosome 3, a region syntenic with human chromosome 3q26.3.

DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication.

The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage lambda in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.

Sulfation and sulfotransferases 1: Sulfotransferase molecular biology: cDNAs and genes.

Sulfotransferase (ST) enzymes catalyze the sulfate conjugation of many hormones, neurotransmitters, drugs, and xenobiotic compounds. These reactions result in enhanced renal excretion of the sulfate-conjugated reaction products, but they can also lead to the formation of "bioactivated" metabolites. ST enzymes are members of an emerging gene superfamily that presently includes phenol ST (PST), hydroxysteroid ST (HSST), and, in plants, flavonol ST (FST) "families," members of which share at least 45% amino acid sequence identity. These families can be further subdivided into "subfamilies" that are at least 60% identical in amino acid sequence. For example, the PST family includes both PST and estrogen ST (EST) subfamilies. Amino acid sequence motifs exist within ST enzymes that are conserved throughout phylogeny. These signature sequences may be involved in the binding of 3'-phosphoadenosine-5 '-phosphosulfate, the cosubstrate for the sulfonation reaction. There are presently five known human cytosolic ST enzymes: an EST, an HSST, and three PSTs. cDNAs and genes for all of these enzymes have been cloned, and chromosomal localizations have been reported for all five genes. Genes for these human enzymes, as well as those of other mammalian cytosolic ST enzymes that have been cloned, show a high degree of structural homology, with conservation of the locations of most intron/exon splice junctions. Human ST enzyme expression varies among individuals. Functionally significant genetic polymorphisms for ST enzymes in humans have been reported, and other molecular genetic mechanisms that might be involved in the regulation of the expression of these enzymes are being explored. Knowledge of the molecular biology of cytosolic ST enzymes, when placed within a context provided by decades of biochemical research, promises to significantly enhance our understanding of the regulation of the sulfate conjugation of hormones, neurotransmitters, and drugs.

Human jejunal estrogen sulfotransferase and dehydroepiandrosterone sulfotransferase: immunochemical characterization of individual variation.

Sulfate conjugation is an important metabolic pathway for many drugs, xenobiotic compounds, and steroid hormones. Human tissues express five cytoplasmic sulfotransferase (ST) enzymes: estrogen ST (EST), dehydroepiandrosterone (DHEA) ST, and three phenol STs (PSTs). Both EST and DHEA ST can catalyze the sulfonation of steroid compounds, including exogenously administered steroids such as ethinyl estradiol. We set out to characterize immunochemically the nature and extent of individual variation in the expression of EST and DHEA ST in the human small intestine after Northern blot analysis had demonstrated that both enzymes were expressed in that tissue. Polyclonal antibodies to human EST and DHEA ST were developed, and Western blot analysis demonstrated that the antibodies were specific. We then performed quantitative Western blots of EST and DHEA ST in 62 samples of human jejunal mucosa. Large individual variations in immunoreactive EST and DHEA ST protein levels were present in those 62 tissue samples. However, there was not a significant correlation between levels of immunoreactive protein for the two enzymes (rs = 0.143, p = 0.262), indicating that EST and DHEA ST in the human jejunum are regulated independently. Furthermore, immunoreactive EST and DHEA ST protein levels in these samples did not differ significantly between the genders, and neither was correlated significantly with time of tissue storage, patient age, or underlying pathology. Frequency distribution histograms of immunoreactive protein values were skewed for both enzymes, and the DHEA ST frequency distribution seemed to be bimodal. These results represent a step toward understanding the molecular basis for individual variation in the expression and function of EST and DHEA ST in the human small intestine.

Human estrogen sulfotransferase gene (STE): cloning, structure, and chromosomal localization.

Sulfation is an important pathway in the metabolism of estrogens. We recently cloned a human liver estrogen sulfotransferase (EST) cDNA. We have now determined the structure and chromosomal localization of the EST gene, STE, as a step toward molecular genetic studies of the regulation of EST in humans. STE spans approximately 20 kb and consists of 8 exons, ranging in length from 95 to 181 bp. The locations of most exon-intron splice junctions within STE are identical to those found in a human phenol ST (PST) gene, STM, and in a rat PST gene. In addition, the locations of five STE introns are also conserved in the human dehydroepiandrosterone (DHEA) ST gene, STD. The 5'-flanking region of STE contains one CCAAT and two TATA sequences. The location of one of the TATA box elements is in excellent agreement with the site of transcription initiation as determined by 5'-rapid amplification of cDNA ends. STE was mapped to human chromosome 4q13.1 by fluorescence in situ hybridization. Cloning and structural characterization of STE will now make it possible to study potential molecular genetic mechanisms involved in the regulation of EST in human tissues.

Human thermolabile phenol sulfotransferase gene (STM): molecular cloning and structural characterization.

Thermolabile (TL) phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic monoamines such as dopamine. The level of activity of TL PST in at least one human tissue, the blood platelet, is controlled by a genetic polymorphism. We previously cloned and expressed a cDNA for human liver TL PST and localized its gene, STM, to human chromosome 16p11.2, a region of the chromosome to which the Batten disease gene is also localized. A cDNA for human brain TL PST with an identical open reading frame (ORF) has also been cloned. We have now isolated the human TL PST gene, STM, and have characterized its structural organization as an additional step toward understanding molecular mechanisms involved in the regulation of levels of TL PST activity in human tissue. STM consists of ten exons and nine introns, with a total length of approximately 8.4 kb. Exons range from 88-499 bp in length, while introns vary from 89-1855 bp. Many of the exon-intron splice junctions in STM are located at positions identical to those of splice junctions in the human dehydroepiandrosterone (DHEA) ST gene, STD, and the rat phenol or aryl ST gene. The first two STM exons are represented in the 5'-UTR of a longer TL PST cDNA expressed in both brain and liver, while exon III is represented in a shorter cDNA 5'-UTR expressed in both tissues. These observations suggest alternative transcription initiation and/or alternative splicing as explanations for the existence of TL PST mRNA species with two different 5'-UTRs. 5'-Flanking region(s) of STM contained neither canonical TATA nor CCAAT elements, but they did contain pyrimidine-rich stretches. Northern blot analyses showed that an mRNA species approximately 1.4 kb in length was expressed in human liver, kidney, lung, small intestine, spleen and leukocyte. Molecular cloning and structural characterization of STM will make it possible to study molecular genetic mechanisms involved in the regulation of TL PST activity in human tissue.

Thermolabile phenol sulfotransferase gene (STM): localization to human chromosome 16p11.2.

Thermolabile (TL) phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic monoamine neurotransmitters such as dopamine and serotonin. We recently cloned a cDNA for human liver TL PST and expressed it in COS-1 cells. We now report the chromosomal localization of the human TL PST gene (STM) as well as its partial sequence. DNA from NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panels 1 and 2 was screened by use of the PCR, and the STM gene was mapped to chromosome 16. Regional localization to 16p11.2 was performed by PCR analysis of a high-resolution mouse/human somatic cell hybrid panel that contained defined portions of human chromosome 16.

Human liver estrogen sulfotransferase: identification by cDNA cloning and expression.

Sulfation is an important pathway in the biotransformation of steroid hormones such as estrogens. Human liver contains three well-characterized sulfotransferase (ST) enzymes, dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Although two of these enzymes, DHEA ST and one form of PST, can catalyze the sulfate conjugation of estrogens, our goal was to test the hypothesis that human liver might also contain a distinct estrogen ST (EST). We used the PCR to clone a human liver EST cDNA by utilizing degenerate oligonucleotide primers designed on the basis of highly conserved EST sequences in non-human species to amplify a 512 nucleotide portion of the open reading frame (ORF) with single-stranded human liver cDNA as a template. Rapid amplification of cDNA ends (RACE) was then used to obtain the 5'- and 3'-ends of the EST cDNA. The ORF of this cDNA consisted of 882 nucleotides and encoded 294 amino acids. The protein encoded by the human liver EST cDNA was 81%, 73%, and 72% identical to the amino acid sequences of guinea pig adrenocortical, bovine placental and rat liver ESTs, respectively. Human liver EST transiently expressed in COS-1 cells was capable of catalyzing the sulfation of estrone, DHEA and 4-nitrophenol, but not that of dopamine. The expressed human liver EST was also characterized with regard to thermal stability, inhibition by 2,6-dichloro-4-nitrophenol (DCNP) and response to NaCl. Identification of human liver EST by cloning and expression of its cDNA should enhance understanding of the enzymology and regulation of the biotransformation of estrogens and other steroids in humans.

Human liver thermolabile phenol sulfotransferase: cDNA cloning, expression and characterization.

The sulfate conjugation of phenolic biogenic amines in humans is catalyzed by the thermolabile (TL) form of phenol sulfotransferase (PST). As a first step toward cloning a cDNA for TL PST, the enzyme was purified from jejunal mucosa, the human tissue with the highest known specific activity, and purified TL PST was subjected to limited proteolysis and amino acid sequencing. The PCR was then performed with human liver cDNA as template and primers designed on the basis of an 18 amino acid stretch of TL PST sequence to obtain a probe for screening cDNA libraries. When cDNA library screening proved unsuccessful, PCR primers were designed based on the nucleotide sequence of a functionally uncharacterized human brain cDNA that had been speculated to represent an aryl sulfotransferase. This brain cDNA encoded the 18 amino acid sequence that we had determined in TL PST. With human liver cDNA as template, these primers amplified a PCR product that contained an 885 nucleotide open reading frame that encoded 295 amino acids--including the 18 amino acids of TL PST sequence. In vitro transcription and translation of this human liver cDNA resulted in the synthesis of a 35.5 kDa protein. The human liver cDNA was also expressed in COS-1 cells, and the biochemical and physical characteristics of the encoded enzyme were identical with those of human liver TL PST. The cloning of a cDNA for human liver TL PST represents an important step toward understanding the molecular basis for the regulation of this enzyme in humans.

Human liver dehydroepiandrosterone sulfotransferase: nature and extent of individual variation.

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of steroid hormones such as DHEA, estrone, and estradiol. As a first step in pharmacogenetic studies of DHEA ST in humans, we measured individual variation in DHEA ST enzymatic activity and thermal stability in 94 samples of human hepatic tissue, 39 of which were from patients with normal liver function studies. Neither level of enzyme activity nor thermal stability were significantly correlated with either time of tissue storage at -80 degrees C or patient age. In addition, there were no gender-dependent differences in DHEA ST activity in these samples. DHEA ST enzymatic activity varied 4.6-fold, with a mean value of 317 +/- 100 units/gm tissue (mean +/- SD) in all samples and 318 +/- 104 units/gm in the subset of 39 samples from patients with normal hepatic function studies. Frequency distributions of DHEA ST activity for both the entire group of 94 samples and the subset of 39 were bimodal, with 25% and 21% included in a high activity subgroup, respectively. The presence of this high activity subgroup was confirmed when data for samples from male and female patients were evaluated separately and when only data for white patients were examined. The existence of a subgroup of subjects with a high level of DHEA ST enzymatic activity in liver and a 4.6-fold range in this activity have implications for individual differences in the sulfate conjugation of endogenous and exogenously administered steroid hormones and raise the possibility of pharmacogenetic regulation of this important enzyme in humans.

Human thiopurine methyltransferase: molecular cloning and expression of T84 colon carcinoma cell cDNA.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. Levels of TPMT activity in human tissue are controlled by a common genetic polymorphism that is an important factor responsible for individual variation in thiopurine drug toxicity and therapeutic efficacy. Our goal was to purify, to obtain a partial amino acid sequence for, and to clone and express cDNA for human TPMT as a first step in determining the molecular basis for this genetic polymorphism. Human kidney TPMT was purified, the protein was subjected to limited proteolysis, and amino acid sequence information was obtained from the resultant peptide fragments. Primers based on the amino acid sequence information were used to amplify a unique sequence from human liver cDNA by use of the polymerase chain reaction. Because TPMT has been reported to be present in the colon, T84 human colon carcinoma cells were studied and were found to express TPMT activity with biochemical properties similar to those of the human kidney and liver enzymes. Oligonucleotide probes based on the human kidney TPMT amino acid sequence were then used to screen a T84 human colon carcinoma cell cDNA library. A 2.7-kilobase cDNA clone was isolated that contained an open reading frame of 735 nucleotides, which encoded a protein of 245 amino acids. The deduced amino acid sequence of the encoded protein included one 24- and two separate 12-amino acid sequences identical to those obtained by sequencing proteolytic fragments of purified human kidney TPMT. Transcripts were made in vitro from the open reading frame of the cDNA clone. These transcripts were translated in a rabbit reticulocyte lysate system, and the resulting translation product comigrated with human kidney TPMT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The T84 cell cDNA clone, truncated within the 3' untranslated region at an Sstl restriction site, was then used to create an expression construct with the eukaryotic expression vector P91023(B), and this construct was used to transfect COS-1 cells. The transfected cells expressed a high level of TPMT enzymatic activity, and this activity displayed a pattern of inhibition by TPMT inhibitors identical to that of human kidney and T84 human colon carcinoma cell TPMT. Cloning of cDNA for this important drug-metabolizing enzyme may make it possible to define the molecular basis of the TPMT genetic polymorphism in humans.

Cholesterol sulfation in human liver. Catalysis by dehydroepiandrosterone sulfotransferase.

Cholesterol can undergo sulfate conjugation to form cholesterol 3-sulfate. Our experiments were performed to determine whether human liver cytosol could catalyze the sulfation of cholesterol, and, if so, whether any of the three well-characterized human hepatic cytosolic sulfotransferases, dehydroepiandrosterone sulfotransferase (DHEA ST), thermostable (TS) phenol sulfotransferase (PST), or thermolabile (TL) PST might participate in the reaction. On the basis of substrate kinetics, two "forms" of cholesterol sulfotransferase (CST) activity were present in human liver cytosol, one with high and one with low affinity for cholesterol. Apparent KM values of the high- and low-affinity activities were 0.14 and 15 microM for cholesterol and 0.30 and 0.19 microM for 3'-phosphoadenosine-5'-phosphosulfate, respectively. Both kinetic forms of CST activity had thermal inactivation profiles similar to those of DHEA ST and TS PST, but both were more thermostable than was TL PST. Enzyme inhibition studies performed with 2,6-dichloro-4-nitrophenol (DCNP) showed that inhibition profiles for both high- and low-affinity CST activities were similar to those of DHEA ST and TL PST, but both were more resistant to DCNP inhibition than was TS PST. Experiments performed with 20 individual human liver samples confirmed these observations and demonstrated highly significant correlations between both high- and low-affinity CST activities and DHEA ST activity (rs = 0.740, p = 0.0001 and rs = 0.767, p < 0.0001, respectively). However, the level of activity of neither kinetic form of CST activity was significantly correlated with either TS or TL PST activities.(ABSTRACT TRUNCATED AT 250 WORDS)

D-3-deoxy-3-substituted myo-inositol analogues as inhibitors of cell growth.

A number of unnatural D-3-deoxy-3-substituted myo-inositols were synthesized and their effects on the growth of wild-type NIH 3T3 cells and oncogene-transformed NIH 3T3 cells were studied. The compounds were found to exhibit a diversity of growth-inhibitory activities and showed selectivity in inhibiting the growth of some transformed cells as compared with wild-type cells. Remarkably, D-3-deoxy-3-azido-myo-inositol exhibited potent growth-inhibitory effects toward v-sis-transformed NIH 3T3 cells but had little effect on the growth of wild-type cells. The growth-inhibitory effects of the myo-inositol analogues were antagonized by myo-inositol. Since [3H]-3-deoxy-3-fluoro-myo-inositol was shown to be taken up by cells and incorporated into cellular phospholipids, we suggest that these unnatural myo-inositol analogues may act as antimetabolites in the phosphatidylinositol intracellular signalling pathways. Because cells transformed by oncogenes often exhibit elevated phosphatidylinositol turnover, the inhibition of signalling pathways that mediate oncogene action could offer new opportunities for controlling the growth of cancer cells.

The effect of naloxone on the blood levels of morphine and histamine in morphine dependent dogs.

Naloxone administration to morphine dependent dogs has caused an increase in the blood level of morphine, which was transient and statistically significant up to 1 min following the injection of the drug, although concomitant increase in the blood histamine level was not observed. Subsequent injection of naloxone has failed to produce an increase in the blood level of morphine. Likewise, injection of naloxone to morphine dependent dogs pretreated with compound 48/80, a potent mast cell depleter, revealed no increase in the blood level of morphine. This was attributed to the fact that, the origin of morphine, released into the blood, was the mast cells.