Heat Transport in Herbertsmithite: Can a Quantum Spin Liquid Survive Disorder?

One favorable situation for spins to enter the long-sought quantum spin liquid (QSL) state is when they sit on a kagome lattice. No consensus has been reached in theory regarding the true ground state of this promising platform. The experimental efforts, relying mostly on one archetypal material ZnCu_{3}(OH)_{6}Cl_{2}, have also led to diverse possibilities. Apart from subtle interactions in the Hamiltonian, there is the additional degree of complexity associated with disorder in the real material ZnCu_{3}(OH)_{6}Cl_{2} that haunts most experimental probes. Here we resort to heat transport measurement, a cleaner probe in which instead of contributing directly, the disorder only impacts the signal from the kagome spins. For ZnCu_{3}(OH)_{6}Cl_{2}, we observed no contribution by any spin excitation nor obvious field-induced change to the thermal conductivity. These results impose strong constraints on various scenarios about the ground state of this kagome compound: while certain quantum paramagnetic states other than a QSL may serve as natural candidates, a QSL state, gapless or gapped, must be dramatically modified by the disorder so that the kagome spin excitations are localized.

Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human alpha class glutathione S-transferase gene.

Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.

Preferential increase of glutathione S-transferase class alpha transcripts in cultured human hepatocytes by phenobarbital, 3-methylcholanthrene, and dithiolethiones.

In rodents, a diversity of compounds are able to protect against acute and chronic toxicities of various xenobiotics including carcinogens, at least in part through induction of drug-metabolizing enzymes including glutathione S-transferase (GST) enzymes. We have posed the question as to whether or not these compounds also induce GSTs in human liver. Primary human hepatocyte cultures were exposed to phenobarbital, 3-methylcholanthrene, and two dithiolethiones [1,2-dithiole-3-thione and its 5-(2-pyrazinyl)-4-methyl derivative, oltipraz], and steady-state mRNA levels of GST classes alpha, mu, and pi were determined by Northern blot analysis. After 3 daily treatments, the two dithiolethiones were the most potent inducers; phenobarbital was also effective but to a lesser extent and 3-methylcholanthrene increased GST mRNA in only 2 of the 6 samples, although it stimulated cytochrome P-450 1A2 mRNA in all cell preparations. Whatever the compound only GSTA1 and/or A2 transcripts were induced. GST M1 mRNAs were not responsive or only slightly responsive, and GST P1 mRNAs, which were mostly undetectable in control cells, were not affected by treatment with any of the four chemicals. Large individual variations were observed in the level of induction of GST A1 and/or A2 mRNAs, and no sex difference could be demonstrated. These results clearly indicate that phenobarbital, 3-methylcholanthrene, and dithiolethiones are able to markedly increase mRNA levels of GST in human hepatocytes and that the GST alpha class is preferentially involved.

3'-end labeling of DNA with [alpha-32P]cordycepin-5'-triphosphate.

Cordycepin-5'-triphosphate (3'-deoxyadenosine-5'-triphosphate) can be incorporated into the 3'-ends of DNA fragments using terminal deoxynucleotidyl transferase from calf thymus (Bollum, 1974). Because cordycepin-5'-monophosphate lacks a 3'-OH group, only a single residue is incorporated. Furthermore, DNA molecules that contain cordycepin-5'-monophosphate at their 3'-ends become resistant to hydrolysis by exonucleases that require free 3'-OH ends. As an alternative to 5'-end labeling of complementary DNA strands, we have used [32P]cordycepin-5'-triphosphate labeling of 3'-ends to confirm the nucleotide sequence of a HhaI-endonuclease-generated pTU4-plasmid DNA fragment that contains several hot spots for insertions of the transposable genetic element Tn3. 3'-End labeling with [32P] cordycepin-5'-triphosphate has also proved useful in determining the sequence of the pTU4 DNA in the vicinity of a strategically located SstII endonuclease cleavage site in the replication region of the plasmid.

High-dose-rate afterloading technique in the radiation treatment of uterine cervical cancer: 399 cases and 9 years experience in Taiwan.

Carcinoma of the uterine cervix has been the leading malignant neoplasm treated in our department. A comparative study was carried out using conventional low dose rate (LDR) with 137Cs sources (less than or equal to 100 cGy/hr) versus high dose rate (HDR) with 60Co sources (greater than 100 cGy/min) in the intracavitary (IC) application following external pelvic irradiation. A total of 399 patients were treated with external RT plus HDR radiation treatment alone from February 1980 through December 1985. Stage IIb and IIIb comprised 79.4% of cases (317 cases). The rate of initial complete response of the tumor, local control, and survival rate seemingly were better in the HDR group, but there was no significant difference statistically. The actuarial survival rates in all cases/definitive RT cases are 85%/85% for Stage 0-IIa, 53%/70% for IIb, 43%/49% for IIIa, 43%/53% for IIIb, 42%/47% for IVa, respectively. Complications were similar and the rectal complications were slightly higher in HDR group. The combination of pelvic irradiation with HDR intracavitary irradiation was more convenient for patients and also for personnel. The HDR technique may be a good substitution for IC treatment.

[Evaluation of quality assurance program in high dose rate remote after-loading brachytherapy].

In the past 9 years, we have routinely used high-dose-rate remote afterloading systems, a Toshiba RAL-302-2 remote afterloading system and a self-fabricated Ir-192 system, for radiation therapy of various cancers, particularly cancers of the uterine cervix and nasopharynx. The frequency of using these two systems is more than 30 times per week. These machines can deliver high-dose radiation (300-1000 cGy) to a specifically planned target on the patient in a few minutes, therefore, the performance of the systems requires well planned dosimetry and quality control assurance in order to achieve effective therapeutic results and ensure safety of the patient under treatment. Any malfunction of the machines may cause over-radiation that exceeds a planned dose to the patient, and any distortion in the distribution of a planned dose may give an unexpected high dose of radiation to the therapist operating the machines. Hence, a quality assurance program must be enforced regularly to avoid or minimize any possible accidents. Based on information and recommendations reported in the literature, as well as from our own experience, we set up a program of routine checks to correct and adjust the machinery conditions and to ensure the optimum operational status of the equipment on a weekly, monthly, semiannual and annual basis. In checking the Co-60 machine, we found that the rate of occurrence for abnormal source position was 3.9% and that for the timer was 2.8%. In the Ir-192 machine checks, abnormal conditions were found only in the source position at the rate of 7.8%.

Implementation of stereotactic focal radiotherapy for intracranial arteriovenous malformations using a linear accelerator.

A system of stereotactic focal radiotherapy using a linear accelerator has been developed in cooperation with a neurosurgeon. The treatment is delivered using a carefully calibrated 10 MV machine and the Cosman-Roberts-Wells (CRW) system. The precision of the method as well as its quality assurance is described. Eight patients with intracranial arteriovenous malformations (AVM) received irradiation from August 1990 to November 1991. The prescribed dose at the periphery of the AVM was 8 Gy per session, with six patients receiving two sessions and two patients receiving one session. The field size, encompassing the 90% isodose, ranged from 20 mm to 35 mm. In four patients, follow-up angiography was performed one year after the full course of therapy; total obliteration of the AVM was noted in three (75%) with a partial response in the other. In the other four patients, follow-up angiography was not performed; one patient, who had only one session of irradiation, experienced rebleeding six months later and died, and the other three patients had no further episodes of bleeding during their follow-up of 28, 18 and 14 months, respectively. Linear accelerator-based stereotactic focal radiotherapy can attain a precisely defined and reproducible dose distribution. The effects of this treatment may take one to two years to develop. Our preliminary study suggests that it is an effective alternative treatment for surgically inaccessible lesions. Patients with a small cavernous sinus dural AVM appear to have a better and more rapid response.

Cloning and sequencing of a cDNA for a ligninase from Phanerochaete chrysosporium.

Lignin is a complex polymer of phenylpropanoid subunits. It is an essential component of woody tissue, to which it imparts structural rigidity. Lignin is remarkably resistant to degradation by most microbes; nevertheless, a few species of white-rot fungi are able to catalyse its oxidation to CO2. Its biodegradation is of great ecological significance because, next to cellulose, lignin is the most abundant renewable polymer on Earth. The first step in lignin degradation is depolymerization, catalysed by the lignin peroxidase isozymes (ligninases). These isozymes are secreted, along with hydrogen peroxide (H2O2) by the fungus Phanerochaete chrysosporium Burds, under conditions of nutrient (nitrogen) limitation. Ligninases are not only important in lignin biodegradation, but are also potentially valuable in chemical waste disposal because of their ability to degrade environmental pollutants. We have undertaken the cloning of the ligninase genes to understand further their regulation and enzymology. We report here the isolation and characterization of a ligninase complementary DNA clone with a full-length insert. The cDNA sequence shows that the sequence of the mature ligninase is preceded by a 28-residue leader, and the mature protein is predicted to have a relative molecular mass of 37,000 (Mr 37K). Consistent with the classification of ligninase as a peroxidase certain residues thought to be essential for peroxidase activity can be identified and near these residues the ligninase shows homology with other known peroxidases. Our cDNA clone has also allowed us to show that expression of ligninase is regulated at the messenger RNA level.

Effect of DNA sequences adjacent to the termini of Tn3 on sequential translocation.

Insertion of Tn3 generates a five base pair repeat of a nucleotide sequence indigenous to the recipient genome. Tn3 promoted deletions extend precisely from the Tn3 terminus and remove one of the 5 base pair repeats while not affecting the ability of Tn3 to subsequently undergo translocation. A direct repeat of a 10 bp sequence located in the Tn3 termini occurs internally within Tn3 and may affect the orientation of insertion.

Drosophila glutathione S-transferases have sequence homology to the stringent starvation protein of Escherichia coli.

The Drosophila glutathione S-transferase D genes encode a family of isozymes. We have determined the amino acid sequence of a new member of this family by nucleotide sequence analysis of a genomic DNA clone. The open reading frame of this intronless gene should encode an isozyme subunit of 211 amino acids. This sequence has significant homology to the E. coli stringent starvation protein, SSP, which is also a protein of two identical 211 amino acid subunits. The two proteins have very similar overall amino acid composition as well. It is possible that SSP may be a glutathione S-transferase(s) in E. coli or is evolutionarily related to glutathione S-transferases. Because SSP is known to be tightly associated with the RNA polymerase holoenzyme during purification, it is conceivable that Drosophila glutathione S-transferase(s) may potentially interact with the transcription machinery in a fashion similar to SSP's interaction with E. coli RNA polymerase holoenzyme.

Suppression of glutathione S-transferases in rat seminal vesicles or pituitary glands.

We have studied the tissue-specific expression of GSH S-transferases in rat seminal vesicles and pituitary glands by in vitro translation and immunoprecipitation. The major GSH S-transferase subunit expressed in rat seminal vesicles belongs to the Yb mobility class whose expression diminishes when the rats are treated with pentobarbital. The pattern of GSH S-transferase expression in the pituitary gland is very similar to that of the rat brain with Yb size subunit(s) predominant. The Y beta size subunit is also expressed together with the Yc and Y delta subunits. The expression of GSH S-transferases was drastically reduced in pituitary gland poly(A) RNAs from diethylstilbestrol-treated, ovariectomized female rats. Xenobiotics such as phenobarbital, 3-methylcholanthrene, and trans-stilbene oxide induce rat liver GSH S-transferase activities, especially the Ya- and Yb-subunit containing isozymes. Induction of GSH S-transferases by a combination of the three xenobiotics is neither additive nor synergistic, however. Our results clearly demonstrate that GSH S-transferase expression in seminal vesicles and pituitary glands can be suppressed by phenobarbital and diethylstilbestrol, respectively. Our findings suggest that different GSH S-transferase isozymes respond differently to various xenobiotics. Both induction and suppression occur in rats treated with xenobiotics. This notion helps to explain the lack of additive or synergistic induction in rats treated with more than one xenobiotic.

Rat glutathione S-transferases supergene family. Characterization of an anionic Yb subunit cDNA clone.

High multiplicity of GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Subunit composition analysis of rat liver GSH S-transferases indicated that heterodimer associations were not random, limiting the generation of GST isozyme multiplicity. We have analyzed a Yb subunit cDNA clone, pGTR187, that may correspond to an anionic Yb subunit sequence. Comparison with other GSH S-transferase cDNA sequences and blot hybridization results indicates that the multiple Yb subunits are encoded by a multigene family. This Yb subunit sequence has very limited homology to Ya and Yc subunit cDNAs, but slightly more sequence homology to the Yp subunit cDNA. More consistent sequence homology is found at the amino acid level with 28% conservation throughout the coding sequences. These results and results published from other laboratories clearly indicate that rat GSH S-transferases are products of at least four different gene families that constitute a supergene family. Conceptually, the supergene family may encode GSH S-transferases of very different structures that are essential to metabolize a multitude of xenobiotics in addition to serving other physiologically important functions.

Complete sequence of IS3.

We have determined the nucleotide sequence of IS3. Our IS3 isolate has 39 bp inverted repeats (IR's) with 6 mismatches, and is 1258 bp long. IS3 contains a large open reading frame (ORF) of 288 codons with a smaller, partially overlapping ORF of 91 codons on the opposite strand in codon-codon register. The large ORF is preceded by and has a 4 bp overlap with a 99 codon ORF that has potential transcriptional and translational start signals. Thus, IS3 could encode a bicistronic mRNA. The Shine-Dalgarno sequence for this 99 codon ORF could be sequestered in a stem-loop structure, but only if the transcript began outside IS3, as was first seen with IS10. This could be a means for preventing fortuitous activation of IS3 by outside promoters. No DNA sequence homology was found between IS3 and other prokaryotic IS elements, but there is slight amino acid sequence homology and significant conservation of hydropathicity patterns between the putative transposases of IS3 and IS2.

tnpM: a novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate resolution.

We have identified a new gene, tnpM, in Tn21 that encodes the 12.6 kilodalton modulator protein. The Tn21 modulator enhances Tn21 transposition and suppresses resolution of cointegrate replicons in vivo. A putative binding site may be located in the N-terminal portion of the TnpR (resolvase) structural gene sequences. Tn501 transposition and cointegrate resolution can be regulated by the subcloned tnpM gene of Tn21 in trans-complementation experiments. Examination of the Tn501 DNA sequence also reveals a potential tnpM coding sequence upstream of the Tn501 resolvase gene. We conclude that Tn21 and Tn501 are different from Tn3 and Tn1000 both in genome organization and in regulation of transposition functions.

On the multiplicity of rat liver glutathione S-transferases.

Rat liver glutathione S-transferases have been purified to apparent electrophoretic homogeneity by S-hexylglutathione-linked Sepharose 6B affinity chromatography and CM-cellulose column chromatography. At least 11 transferase activity peaks can be resolved including five Yb size homodimeric isozymes, two Yc size homodimeric isozymes, one Ya homodimeric isozyme, one Y alpha homodimeric isozyme, and two Ya-Yc heterodimeric isozymes. Distribution of the GSH peroxidase activity among the CM-cellulose column fractions suggests the existence of further multiplicity in this isozyme family. Substrate specificity patterns of the Yb subunit isozymes revealed a possibility that each of the five Yb-containing isozymes is composed of a different homodimeric Yb size subunit composition. Our findings on the increasing multiplicity of glutathione S-transferase isozymes are consistent with the notion that multiple isozymes of overlapping substrate specificities are required to detoxify a multitude of xenobiotics in addition to serving other important physiological functions.

Human liver glutathione S-transferases: complete primary sequence of an Ha subunit cDNA.

Multiple human liver GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Human liver GSTs are composed of at least two classes of subunits, Ha (Mr = 26,000) and Hb (Mr = 27,500). Immunological cross-reactivity and nucleic acid hybridization studies revealed a close relationship between the human Ha subunit and rat Ya, Yc subunits and their cDNAs. We have determined the nucleotide sequence of the Ha subunit 1 cDNA, pGTH1. The alignments of its coding sequence with the rat Ya and Yc cDNAs indicate that they are approximately 80% identical base-for-base without any deletion or insertion. Regions of sequence homology (greater than 50%) have also been found between pGTH1 and a corn GST cDNA and rat GST cDNAs of the Yb and Yp subunits. Among the 62 highly conserved amino acid residues of the rat GST supergene family, 56 of them are preserved in the Ha subunit 1 coding sequences. Comparison of amino-acid replacement mutations in these coding sequences revealed that the percentage divergence between the rat Ya and Yc genes is more than that between the Ha and Ya or Ha and Yc genes.

Biochemical characterization of Drosophila glutathione S-transferases D1 and D21.

The genomic DNA for the two Drosophila genes, gstD1 and gstD21, were engineered for expression in Escherichia coli by polymerase chain reaction using a pair of specially designed primers. This newly designed expression system produced consistently high yields of the recombinant glutathione S-transferases (GSTs), which were purified to electrophoretic homogeneity by S-hexyl-GSH affinity chromatography. Consistent with their differences in size, GST D1 and GST D21 displayed different mobilities on SDS-polyacrylamide gel electrophoresis. Circular dichroism spectrometry revealed some differences in the protein secondary structural organization between the two GST D isozymes. Polyclonal antibodies against GST D1 and GST D21 revealed that they are immunologically distinct from each other. The GST D1 antiserum cross-reacted weakly with GST D21, but the GST D21 antiserum had no detectable cross-reactivity with GST D1. The amino acid sequences of GST D1 and GST D21 have 70% identity. GST D1 is active toward CDNB with 17% of the catalytic efficiency of the human alpha GST121, whereas CDNB is a poor substrate for GST D21. Both GST D1 and GST D21 have similar levels of GSH peroxidase activity against cumene hydroperoxide. Another major difference in substrate specificities between GST D1 and GST D21 is in the activity of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) dehydrochlorinase, which exists only in the GST D1 isozyme. This is the first definitive demonstration that DDT dehydrochlorinase activity is an intrinsic property of a Drosophila GST. Our results suggest that GST D1 may play a role in DDT metabolism in Drosophila.

Pentobarbital-induced changes in Drosophila glutathione S-transferase D21 mRNA stability.

The Drosophila glutathione S-transferase (gstD) genes are a family of divergently transcribed, intronless genes and pseudogenes. Under control conditions, the steady-state level of gstD1 mRNA is 20-fold higher than that of the gstD21 mRNA despite a lower transcription rate of the gstD1 gene. The GST D1 protein level is four times as abundant as the GST D21 protein. The gstD1 and gstD21 genes responded rapidly to pentobarbital (PB) as changes in mRNA levels were detectable within 30 min of treatment. Maximal induction of gstD1 and gstD21 resulted in 3-fold and 20-fold elevation of their respective mRNA levels. The major mechanism for the increase in gstD1 mRNAs appears to be transcriptional activation. The 2-fold increase in the rate of gstD21 transcription, however, cannot fully account for the 20-fold increase in the steady-state level of gstD21 mRNA. Therefore, post-transcriptional mechanism(s) should also be responsible for the increase of gstD21 mRNA by PB. Because the gstD21 mRNA is relatively unstable under control conditions, induction of the intronless gstD21 mRNA by PB occurs mainly at the level of enhanced mRNA stability. The GST D1 protein level in adult Drosophila was increased approximately 2-fold after PB treatment, whereas the GST D21 level remained relatively the same. Thus, an increase in gstD21 mRNA stability by PB treatment is probably coupled to a regulatory effect at the translational level.

Drosophila glutathione S-transferase D27: functional analysis of two consecutive tyrosines near the N-terminus.

The Drosophila glutathione S-transferase D27 (GST D27) has been purified and characterized after direct expression of the intronless gstD27 gene in E. coli. The GST D27 has both conjugation activity against the common substrate 1-chloro-2,4-dinitrobenzene and peroxidase activity against cumene hydroperoxide. Its pH optimum is 8.5 in 0.125 M bis-tris propane buffer at 22 degrees C. It is more thermal labile than the human GST121. The GST D27 has two tyrosines at positions 3 and 4. Both of them appear to be important but neither of them is essential for the enzyme activity. Thus, other residues may also participate in catalysis. The two tyrosines of GST D27 could also be important in binding to GSH or S-hexyl GSH. Results from in vitro biochemical analyses were confirmed by the in vivo activity-based CDNB growth inhibition analyses. Our results clearly indicate that the Drosophila GST D isozymes are different from any of the known mammalian GSTs.