Cloning and sequence analysis of cDNA coding for a lectin from Helianthus tuberosus callus and its jasmonate-induced expression

Two lectins (designated as HTA I and HTA II) that seemed to be isolectins were found in Helianthus tuberosus callus. cDNA encoding HTA I was isolated from a ZAP Express expression library by immunoselection by using the anti-HTA antiserum. The sequence of this cDNA consisted of 432 bp nucleotides coding for a polypeptide of 143 amino acid residues (Mr, 15,314). When introduced into E. coli, the cDNA directed the synthesis of active HTA I as indicated by the hemagglutination activity. The deduced amino acid sequence showed homology with some lectins and jasmonate-induced proteins. When callus was cultured in the presence of methyl jasmonate (MeJA), the hemagglutination activity increased in a dose-dependent manner. The levels of expression of the HTA protein and of the corresponding mRNA also increased in the treated callus. In view of these results, HTA I is considered to be a jasmonate-induced protein.     

Stimulated Accumulation of Lectin mRNA and Stress Response in Helianthus tuberosus Callus by Methyl Jasmonate

Callus from Helianthus tuberosus expresses a mannose-specific lectin (HTA). The level of HTA mRNA significantly increased one hour after treatment of the callus with 20 mg/l methyl jasmonate. Following this, fragmentation of the callus DNA at regular intervals was observed together with strong self-fluorescence emission in the callus cells.     

Delineation of three subsets of class I human T antigens (HTA) on Molt-4 cells: serologic and regulatory relationship to HLA class I antigens

Three subsets of class I human T antigens (HTA) were serologically identified on the surface of the Molt-4 T lymphoma cell line. The HTA 1 subset is defined by NAI/34, D47, or 10H3.9 cross-reactive m.Ab. and by BL6 m.Ab. The HTA 2 and HTA 3 subsets are defined by M241 and 4A7.6 m.Ab., respectively. We obtained no evidence of any additional HTA subset. The different HTA antigens share only few epitopes with human leukocyte antigens (HLA-A, -B, and -C). Interestingly, these epitopes all belong to the same cluster defined on HLA class I molecules, but differ from one HTA subset to another. These results would therefore suggest that HTA and HLA class I antigens display a limited structural homology, but have a conserved epitopic area whose detailed structure differs for each HTA subset. Furthermore, the cell surface expression of each HTA class I molecule type is differently enhanced by natural interferon (IFN)-alpha or -gamma. This result additionally supports the serologic delineation of HTA subsets, and suggests that the corresponding genes in Molt-4 cells, are subjected to distinct regulations.     

Somatic variants of the level of expression of a cell surface antigen

Somatic variants with constitutive changes in the expression of the cortical thymocyte differentiation antigen HTA 1 were derived from the T-cell leukemia line Molt 4 using monoclonal antibody NA1/34 and the fluorescent activated cell sorter. Cells with the highest and lowest fluorescence were sorted and expanded. After several cycles, high expressor variants, with 10- to 12-fold increased surface HTA 1 and low expressors, with a level of one third relative to the wild-type, were obtained. The stability of the high expressors was improved by cloning. In spite of the large differences in the level of HTA 1 between the various mutants and the wild-type, the expression of HLA-A,B,C or its increase following interferon-alpha stimulation, remained unchanged. Although in HTA 1 there was only a trace of beta2-microglobulin (beta2m) detected by lactoperoxidase labelling of the high expressor variants, significant levels of both beta2m and HTA 1 light chain beta(t) were observed in gels stained with Coomassie blue. The physiological association of beta(t) with HTA 1 was confirmed by the substantial increase of beta(t) which parallels the increase of HTA 1.     

Characterization of a monoclonal antibody, HTA28, recognizing a histone H3 phosphorylation site as a useful marker of M-phase cells.

Mitosis is a valuable indicator of active tissue proliferation but, other than morphological characteristics, there have hitherto been no markers available to detect only M-phase cells. However, a newly established monoclonal antibody (MAb), HTA28, recognizing histone H3 (H3) harboring phosphoserine 28, allows visualization with mitotic chromosomal condensation. In this study we investigated the use of HTA28 for immunohistochemical (IHC) detection of M-phase cells in the regenerating rat liver after partial hepatectomy (PH). Groups of three to five rats were sacrificed at intervals up to 72 hr after PH and proliferation was then assessed by IHC staining using HTA28 and other markers. The temporal pattern of the HTA28 staining index (SI) was very similar to that for the mitotic index (MI), also showing similarities to the bromodeoxyuridine (BrdU) labeling index (LI) with a time lag. The HTA28 SI proved to be higher than MI at every time point in line with HTA28 immunoreactivity maintained for all stages of M-phase. The spatial distribution of HTA28-positive cells corresponded with those of other proliferative cell markers. These therefore provide strong evidence for the applicability of HTA28 as an M-phase marker. We also showed that antigenicity for HTA28 is lost if tissue is not immediately fixed after sampling.     

Progression into the First Meiotic Division Is Sensitive to Histone H2a-H2b Dimer Concentration in Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae contains two genes for histone H2A and two for histone H2B located in two divergently transcribed gene pairs: HTA1-HTB1 and HTA2-HTB2. Diploid strains lacking HTA1-HTB1 (hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2) grow vegetatively, but will not sporulate. This sporulation phenotype results from a partial depletion of H2A-H2B dimers. Since the expression patterns of HTA1-HTB1 and HTA2-HTB2 are similar in mitosis and meiosis, the sporulation pathway is therefore more sensitive than the mitotic cycle to depletion of H2A-H2B dimers. After completing premeiotic DNA replication, commitment to meiotic recombination, and chiasma resolution, the hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2 mutant arrests before the first meiotic division. The arrest is not due to any obvious disruptions in spindle pole bodies or microtubules. The meiotic block is not bypassed in backgrounds homozygous for spo13, rad50Δ, or rad9Δ mutations, but is bypassed in the presence of hydroxyurea, a drug known to inhibit DNA chain elongation. We hypothesize that the deposition of H2A-H2B dimers in the mutant is unable to keep pace with the replication fork, thereby leading to a disruption in chromosome structure that interferes with the meiotic divisions.     

Cytologic features of hyalinizing trabecular adenoma of the thyroid

OBJECTIVE: To clarify the cytologic findings of hyalinizing trabecular adenoma (HTA) in order to reduce erroneous diagnoses of papillary carcinoma. STUDY DESIGN: Review of aspiration cytologic smears of 16 HTA cases and comparison with those of 20 papillary carcinoma cases. RESULTS: The smears from HTA were slightly cellular, and 5 of 16 cases were insufficient for evaluation. Vague, curved nuclear palisading, radiating arrangement surrounding hyaline materials and yellow bodies were observed in 9 (81.8%) of 11 cases that had sufficient material. The tumor cells were mainly spindled; elongated, polygonal and stellate cells were also seen. In 9 of 11 cases, tumor cells with cytoplasmic processes were occasionally observed. The cytoplasm was faintly stained and somewhat filamentous. The cell border was indistinct. Neither papillary nor follicular structures were seen. Intranuclear cytoplasmic inclusions were identified in 100% of HTA and 75% of papillary carcinomas. The incidences of nuclear grooves in HTA and papillary carcinoma were 81.8% and 100%, respectively. CONCLUSION: Cytologic findings indicating HTA are vague, curved nuclear palsiading; radiating arrangement surrounding hyaline material; elongated cells; cell processes; ill-defined cell border; faintly stained and filamentous cytoplasm; yellow bodies; and hyaline material in the background. All are useful cytologic characteristics in distinguishing HTA from papillary carcinoma. A lack of papillary architecture and sheetlike arrangement may also suggest HTA rather than papillary carcinoma.     

卫生技术评估研究结果产出方式的偏好分析

目的 比较研究人员和决策人员对卫生技术评估研究结果产出方式的偏好程度的差异,为扩大卫生技术评估研究的决策使用提供依据。方法 采用问卷调查的方法,收集研究人员和决策人员对不同卫生技术评估研究结果产出方式的评价,运用统计描述的方法,对偏好差异进行描述分析。结果 研究人员和决策人员对卫生技术评估研究结果的产出方式偏好有所差异;在不同行政单位、不同行政级别以及不同教育程度的决策者之间,对产出方式的偏好也存在不同;而不同职称、不同教育程度的研究者,所偏好的成果产出方式则较为一致。结论 进一步强化研究方与决策方之间的沟通交流,根据不同决策人员的需求采取合适的产出方式,加强机构对卫生技术评估结果决策转化的支持,逐步完善激励机制,并强化卫生技术评估结果向广大社会公众的传播。     

Healthcare Databases in Thailand and Japan: Potential Sources for Health Technology Assessment Research

Background

Health technology assessment (HTA) has been continuously used for value-based healthcare decisions over the last decade. Healthcare databases represent an important source of information for HTA, which has seen a surge in use in Western countries. Although HTA agencies have been established in Asia-Pacific region, application and understanding of healthcare databases for HTA is rather limited. Thus, we reviewed existing databases to assess their potential for HTA in Thailand where HTA has been used officially and Japan where HTA is going to be officially introduced.

Method

Existing healthcare databases in Thailand and Japan were compiled and reviewed. Databases’ characteristics e.g. name of database, host, scope/objective, time/sample size, design, data collection method, population/sample, and variables were described. Databases were assessed for its potential HTA use in terms of safety/efficacy/effectiveness, social/ethical, organization/professional, economic, and epidemiological domains. Request route for each database was also provided.

Results

Forty databases– 20 from Thailand and 20 from Japan—were included. These comprised of national censuses, surveys, registries, administrative data, and claimed databases. All databases were potentially used for epidemiological studies. In addition, data on mortality, morbidity, disability, adverse events, quality of life, service/technology utilization, length of stay, and economics were also found in some databases. However, access to patient-level data was limited since information about the databases was not available on public sources.

Conclusion

Our findings have shown that existing databases provided valuable information for HTA research with limitation on accessibility. Mutual dialogue on healthcare database development and usage for HTA among Asia-Pacific region is needed.     

Genomic characterization of thermophilic Geobacillus species isolated from the deepest sea mud of the Mariana Trench

The thermophilic strains HTA426 and HTA462 isolated from the Mariana Trench were identified as Geobacillus kaustophilus and G. stearothermophilus, respectively, based on physiologic and phylogenetic analyses using 16S rDNA sequences and DNA–DNA relatedness. The genome size of HTA426 and HTA462 was estimated at 3.23–3.49 Mb and 3.7–4.49 Mb, respectively. The nucleotide sequences of three independent -phage inserts of G. stearothermophilus HTA462 have been determined. The organization of protein coding sequences (CDSs) in the two -phage inserts was found to differ from that in the contigs corresponding to each insert assembled by the shotgun clones of the G. kaustophilus HTA426 genome, although the CDS organization in another insert is identical to that in the HTA426 genome.     

Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced.     

Smooth muscle cell injury after cryopreservation of human thoracic aortas

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.     

A single amino acid change is responsible for evolution of acyltransferase specificity in bacterial methionine biosynthesis

Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 A resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.     

Renin-angiotensin system polymorphisms in relation to hypertension status and obesity in a Tunisian population

Essential hypertension (HTA) is the clinical expression of a disordered interaction between the genetic, physiological, and biochemical systems that under usual conditions maintain cardiovascular homeostasis. We studied the effects of the angiotensinogen M235T, angiotensin converting enzyme insertion/deletion (ACE I/D), and angiotensin II receptor 1 (AT1R) A1166C gene polymorphisms on the risk of HTA and to evaluate the relationship between these polymorphisms and obesity. We performed AGT, ACE and AGTR genotyping in 142 hypertensive patients and 191 control subjects using PCR-RFLP methods and PCR, respectively. The three polymorphisms were significantly associated with HTA. Individuals carrying the mutated TT of AGT, DD of ACE and CC of AT1R genotypes had an 1.67 (P = 0.032), 3.09 (P < 0.001) and 3.45 (P < 0.001)-fold increased risk of HTA. After adjustment for sex, smoking, diabetes, dyslipidemia, BMI, triglycerides and DD, TT and CC genotypes, BMI was independent risk factor of HTA (OR = 3.14; P < 0.001). An association of BMI with ACE gene polymorphism (P = 0.035), whereas no association with AGT and AT1R gene polymorphisms was obtained. The proportion of hypertensives is as high as 21.8 and 13.4% in the overweight and the obese DD group. The present study implies that the genotyping for the variants of RAS gene could in the future become an important part of the clinical process of risk identification for HTA.     

Genetic transformation of Geobacillus kaustophilus HTA426 by conjugative transfer of host-mimicking plasmids

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, 10(-5)-10(-3) recipient(-1)). pSTE33T showed lower efficiency (10(-7)-10(-6) recipient(-1)) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.     

Involvement of Toll-like receptor 4 and Fc receptors gamma in human neutrophil priming by endotoxins from Escherichia coli

By using the fMLP-induced respiratory burst approach, the involvement of Toll-like receptor 4 (TLR4) in human neutrophil priming by S- or Re-glycoforms of endotoxin from Escherichia coli has been elucidated. The priming effect of Re-glycoform is more pronounced than that of the S-glycoform. Unexpectedly, fMLP-triggered generation of reactive oxygen species (ROS) by endotoxin primed neutrophils was amplified by preincubation of the cells with anti-TLR4 (HTA125) antibodies or with isotype-matched immunoglobulin IgG2a. The most significant finding of our study is that neutrophils exposed to anti-TLR4 antibodies retain their ability to distinguish between S- or Re-glycoforms being primed, respectively. Moreover, differentiated effect of HTA125 antibodies on functional responses of neutrophils during their priming and fMLP stimulation was revealed. Taking these results into consideration, it is reasonable to assume that there is a contribution of Fcγ receptors to fMLP-induced ROS generation by neutrophils preincubated with HTA125 or IgG2a and primed by endotoxins.     

Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans

Homoserine O-acetyltransferase (HTA, EC 2.3.1.31) initiates methionine biosynthesis pathway by catalyzing the transfer of acetyl group from acetyl-CoA to homoserine. This study reports the crystal structure of HTA from Leptospira interrogans determined at 2.2 Å resolution using selenomethionyl single-wavelength anomalous diffraction method. HTA is modular and consists of two structurally distinct domains—a core α/β domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to α/β hydrolase superfamily with the characteristic ‘catalytic triad’ residues in the active site. Detailed structure analysis showed that the catalytic histidine and serine are both present in two conformations, which may be involved in the catalytic mechanism for acetyl transfer.