A patient with primary biliary cirrhosis associated with autoimmune hemolytic anemia.

Primary biliary cirrhosis is often associated with autoimmune conditions, such as thyroid disease, sicca complex, and rheumatoid arthritis. However, an association with autoimmune hemolytic anemia has rarely been reported. We present a case of primary biliary cirrhosis associated with warm type autoimmune hemolytic anemia, and we review prior reports.

Growth suppression of hamster flank organs by topical application of catechins, alizarin, curcumin, and myristoleic acid.

Hamster flank organ growth, as measured by an increase in the area of the pigmented macule, is androgen-dependent. When flank organs of a castrated hamster are treated topically with testosterone, the flank organ becomes larger and darker. Since this growth is known to be dependent on the intracellular active androgen, 5alpha-dihydrotestosterone (DHT), inhibitors of 5alpha-reductase which converts testosterone to DHT can inhibit the growth of the flank organ. Certain unsaturated aliphatic fatty acids, such as gamma-linolenic acid and myristoleic acid, as well as other natural compounds, including alizarin and curcumin, are 5alpha-reductase inhibitors and inhibited flank organ growth. Green tea catechins, including (-)-epicatechin-3-gallate, and (-)-epigallo-catechin-3-gallate (EGCG) are also 5alpha-reductase inhibitors and inhibited flank organ growth. However, (-)-epicatechin and (-)-epigallocatechin, which are not 5alpha-reductase inhibitors, also inhibited flank organ growth. EGCG also inhibited DHT-dependent growth of flank organs. These catechins, therefore, may act by a mechanism other than inhibition of 5alpha-reductase. The effect of EGCG and other compounds was localized at the site of application; they did not affect the growth of the contralateral flank organ in the same animal. Since these compounds do not appear to exhibit systemic effects, they may be potentially useful for treatment of androgen-dependent skin disorders.

Properties and structure of spermidine acetyltransferase in Escherichia coli.

Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion.

An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation.

The polyamine spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine; "glycine-dependent" stimulation, which is due to an increase in the affinity of the receptor for glycine; and voltage-dependent block. These effects may involve three separate polyamine binding sites on the receptor. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD, a polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339-E342) to neutral amino acids reduced or abolished glycine-independent stimulation by spermine without affecting glycine-dependent stimulation or voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg2+. Residue E342 in NR1 appears to be critical for glycine-independent spermine stimulation. Mutations at equivalent positions in NR2A(E352Q) or NR2B(E353Q) had no effect on sensitivity to spermine, pH, or ifenprodil. Residue E342 in NR1 may form part of a discrete spermine binding site on the NMDA receptor or be involved in the mechanism of modulation by polyamines. This residue may also be involved in modulation by protons and ifenprodil.

Correlation between the inhibition of cell growth by accumulated polyamines and the decrease of magnesium and ATP.

The mechanism of the antiproliferation effect of spermidine and spermine was studied using a cell culture system of mouse FM3A cells. The addition of either 10 mM spermidine or 2 mM spermine to the growth medium containing 0.9 mM Mg2+ greatly inhibited cell growth (more than 90%). A decrease in the Mg2+ concentration to 50 microM in the growth medium, but without the polyamine addition, did not influence cell growth. However, the concentrations of spermidine and spermine necessary for the inhibition of cell growth when cells were cultured in the presence of 50 microM Mg2+ were much smaller (2 mM spermidine and 0.15 mM spermine). Nevertheless, the amount of polyamines accumulating in cells which could cause the inhibition of cell growth was almost the same, regardless of the large difference in the added polyamine concentrations. At the early stage of polyamine accumulation, the inhibition of cell growth correlated with the decrease of Mg2+ content, but not with a decrease of the ATP content. The decrease in Mg2+ content correlated well with the inhibition of macromolecular synthesis, especially protein synthesis. Thus, the inhibition of cell growth at the early stage of polyamine accumulation was thought to be due to the inactivation of ribosomes through the replacement of Mg2+ on magnesium-binding sites by polyamines. The decrease in Mg2+ content was mainly caused by the inhibition of Mg2+ transport by polyamines. At the later stage of polyamine accumulation, a decrease in ATP content was also observed. This was followed by swelling of the mitochondria, which may be a symptom of the subsequent cell death.

Spermine-like functions of N1, N12-bis(ethyl)spermine: stimulation of protein synthesis and cell growth and inhibition of gastric ulceration.

The spermine analogue N1, N12-bis(ethyl)spermine (BESPM) could mimic the functions of spermine in the following aspects: 1) BESPM could stimulate globin and ornithine decarboxylase synthesis in a rabbit reticulocyte cell-free system; 2) the addition of BESPM to the culture medium could recover cell growth of polyamine-deficient bovine lymphocytes; 3) spermidine uptake by bovine lymphocytes was inhibited by BESPM and spermine to a comparable degree; and 4) stress-induced gastric ulceration was inhibited by subcutaneous administration of BESPM. Since BESPM was less toxic than spermine for mice, BESPM or its derivatives may be useful for diseases which can be cured by polyamines.

Mechanism of the inhibition of cell growth by N1,N12-bis(ethyl)spermine.

The mechanism of the antiproliferation effect of N1,N12-bis(ethyl)spermine (BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in several aspects [Igarashi, K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A. (1990) Biochem. Biophys. Res. Commun. 172, 715-720]. Our results indicate that not only the decrease in sperimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine-deficient cells caused by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, the behavior of polyamine-deficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N1-acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM-treated cells; thymidine kinase activity did not decrease in BESPM-treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.

Decrease in cell viability due to the accumulation of spermidine in spermidine acetyltransferase-deficient mutant of Escherichia coli.

Physiological functions of spermidine acetyltransferase in Escherichia coli have been studied using the spermidine acetyltransferase (speG) gene-deficient mutant CAG2242 and the cloned speG gene. The growth of E. coli CAG2242 in the defined M9 medium was normal in the presence and absence of 0.5mM spermidine. However, cell viability of E. coli CAG2242 at 48 h after the onset of growth decreased greatly by the addition of 0.5 mM spermidine. The amount of spermidine accumulated in the cells was approximately 3-fold that in the cells grown in the absence of spermidine. Transformation of the cloned speG gene to E. coli CAG2242 recovered the cell viability. Decreased in cell viability of E. coli CAG2242 was observed even when 0.5mM spermidine was added at 24 h after the onset of growth. The results indicate that accumulated spermidine functions at the late stationary phase of growth. The accumulation of spermidine caused a decrease in protein synthesis but not in DNA and RNA synthesis at 28 h after the onset of growth. The synthesis of several kinds of proteins was particularly inhibited. They included ribosome modulation factor and OmpC protein. Since the ribosome modulation factor is essential for cell viability at the stationary phase of growth (Yamagishi, M., Matsushima, H., Wada, A., Sakagami, M., Fujita, N., and Ishihama, A. (1993) EMBO J. 12, 625-630), the decrease in the protein was thought to be one of the reasons for the decrease in cell viability. The decrease in the ribosome modulation factor mainly occurred at the translational level.

Enhancement of cell death due to decrease in Mg2+ uptake by OmpC (cation-selective porin) deficiency in ribosome modulation factor-deficient mutant.

Ribosome modulation factor (RMF) is involved in stabilization of ribosomes during the transition from exponential growth to the stationary growth phase in Escherichia coli. A deficiency of RMF is known to reduce cell viability. Overaccumulation of spermidine also leads to a decrease in cell viability and to a decrease in the synthesis of RMF and of the cation-selective porin OmpC. Thus, a decrease in RMF levels may be involved in the decreased cell viability caused by excess spermidine. Because spermidine also influences the expression of OmpC, we examined whether OmpC deficiency enhances the cell death caused by RMF deficiency. The ompC mutant by itself did not affect protein synthesis or cell viability, but the double rmf ompC mutant produced a much larger decrease in protein synthesis and cell viability than did the single rmf mutant. There was also a decrease in the amount of ribosomes and in the Mg2+ content in the double rmf ompC mutant, and cell viability could be partially restored by the addition of Mg2+ to the growth medium. RMF deficiency was found to inhibit the synthesis of another cation-selective porin OmpF. Thus, the double rmf ompC mutant is deficient in both OmpC and OmpF, which probably accounts for the pronounced decrease in Mg2+ uptake in this mutant. The results indicate that both RMF and Mg2+, acting through stabilization of ribosomes, are important for cell viability at the stationary growth phase.

An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons.

To study the role of acidic residues in modulation of NMDA receptors by spermine, we used site-directed mutagenesis of receptor subunits and voltage-clamp recording in Xenopus oocytes. Sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutated. This region of NR1A shows homology with bacterial amino acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine (D669A), or glutamate (D669E) abolished the "glycine-independent" form of spermine stimulation in heteromeric NR1A/NR2B receptors. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at the equivalent position (D690) in NR1B, which contains the insert encoded by exon 5, reduced the pH sensitivity of NR1B/NR2B receptors. Thus, the effects of mutations at D669 are not prevented by the presence of exon 5, and the influence of exon 5 is not prevented by mutations at D669 (D690 in NR1B). Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg2+ or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine at NR1A/NR2 receptors. Mutations in NR2B at a position (D668) equivalent to D669 did not alter spermine stimulation or sensitivity to pH and ifenprodil. However, mutations D668N and D668A but not D668E in NR2B reduced voltage-dependent block by spermine. Screening of the negative charges at NR1A(D669) and NR2B(D668) may be involved in voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor. Alternatively, this residue may be critical for coupling of modulators such as spermine, protons, and ifenprodil to channel gating.

A regulatory domain (R1-R2) in the amino terminus of the N-methyl-D-aspartate receptor: effects of spermine, protons, and ifenprodil, and structural similarity to bacterial leucine/isoleucine/valine binding protein.

There are complex interactions between spermine, protons, and ifenprodil at N-methyl-D-aspartate receptors. Spermine stimulation may involve relief of proton inhibition, whereas ifenprodil inhibition may involve an increase in proton inhibition. We studied mutations at acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in Xenopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, reduced spermine stimulation and proton inhibition. Mutation NR1(D130N) reduced sensitivity to ifenprodil by more than 500-fold, but had little effect on sensitivity to spermine and pH. Mutations at six other residues in this region of the NR1 subunit reduced the potency and, in some cases, the maximum effect of ifenprodil. These mutants did not affect sensitivity to pH, glutamate, glycine, or other hallmark properties of N-methyl-D-aspartate channels such as Mg2+ block and Ba2+ permeability. Residues in this region presumably form part of the ifenprodil-binding site. To model this region of NR1 we compared the predicted secondary structure of NR1 (residues 19-400) with the known structures of 1,400 proteins. This region of NR1 is most similar to bacterial leucine/isoleucine/valine binding protein, a globular amino acid binding protein containing two lobes, similar to the downstream S1-S2 region of glutamate receptors. We propose that the tertiary structure of NR1(22-375) is similar to leucine/isoleucine/valine binding protein, containing two "regulatory" domains, which we term R1 and R2. This region, which contains the binding sites for spermine and ifenprodil, may influence the downstream S1 and S2 domains that constitute the glycine binding pocket.