The first report of the thyroid function of haemophilic patients with HIV/HCV co-infection in Japan.

INTRODUCTION: A high incidence of thyroid dysfunction is reported in patients with HIV or HCV mono-infection. We have conducted a periodic medical examination including the thyroid function for haemophilic patients with HIV/HCV co-infection due to contaminated blood products. METHODS: We examined the thyroid function (as assessed by the FT3, FT4 and TSH levels) in 45 haemophilic patients, including thyroglobulin and auto-antibody, antithyroglobulin antibody, antithyroid peroxidase antibody and anti-TSH receptor antibody in 28 patients. RESULTS: All the patients were males (median age: 42 years; range: 29-66). The median values of thyroid function were FT3 3.36 pg mL(-1) , FT4 1.125 ng mL(-1) and TSH 1.65 µIU mL(-1) . Five patients (11.1%) had high TSH levels. In 28 patients in whom the presence of auto-antibodies was examined, the median age was 47 years of age. The median value of thyroglobulin was 16 ng mL(-1) and two patients showed high levels of thyroglobulin. The presence of anti-TSH receptor antibody of all the patients was negative, but one patient (3.5%) was positive of antithyroid peroxidase antibody and antithyroglobulin antibody. CONCLUSIONS: Since 0.68-3.6% of the general healthy population is reported to show hypothyroidism, our data showed that the proportion of hypothyroidism in haemophilic patients with HIV/HCV co-infection was more frequent than that of the normal population.

Evaluation of the operative methods for Graves' disease.

AIM: In Japan, surgery for Graves' disease (GD), which is considered to be a radical therapy, has been restricted by various guidelines. Nevertheless, some patients benefit from surgery. We sought to identify a reasonable operative method for GD by comparing the efficacy and safety among patients undergoing different extents of thyroidectomy. METHODS: A total of 162 patients underwent thyroidectomy for GD between 2003 and 2012 in our department. We compared the clinical factors among those who underwent subtotal thyroidectomy (ST), near-total thyroidectomy (NTT), and total thyroidectomy (TT). RESULTS: The ST, NTT, and TT groups included 111, 21, and 30 patients, respectively. The patient sex, period between disease onset and surgery, and preoperative thyroidal function were not substantially different among the three groups. With regard to surgical variables, the duration of surgery, amount of blood loss, and postoperative length of hospitalization were not substantially different among the three groups. Postoperative recurrent laryngeal nerve (RLN) palsy was transient in all cases, but the rate was significantly higher in the TT group compared to the other two groups (P<0.001). The incidences of transient hypocalcemia and permanent hypoparathyroidism were not substantially different among the groups. The proportion of patients who required the postoperative administration of levothyroxine was significantly lower in the ST group compared to the TT and NTT groups. Hyperthyroidism recurrence was noted in eight patients in the ST group (7.2%). CONCLUSION: NTT for GD is thus considered to be a reasonable operative method regarding both efficacy and safety.

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

[Metabolic syndrome].

About 20 years ago, Reaven presented the concept that a series of related factors such as hyperinsulinemia, hypertension, low high-density lipoprotein (HDL)-cholesterol levels, and hypertriglyceridemia tended to co-occur in the same individual and that this risk-factor clustering and its association with insulin resistance might be of critical importance in the underlying cause of cardiovascular disease. This risk-factor clustering called as "syndrome X" has now become "metabolic syndrome" (METS). Nowadays, METS is becoming well known as a condition of high-risk for the subsequent development of ischemic cardiovascular disease in Western population as well as Japanese population and it is proved that the prevalence of METS is very common. There are several different diagnostic definitions for METS. In Japanese definition, waist circumference is essential for criterion of METS because visceral fat accumulation is believed to be associated with METS more closely than the body mass index (BMI) itself or the amount of subcutaneous fat. Therefore treatment strategy to reduce visceral fat seems to be crucial for management of patients with METS. Adipose tissue is not simply an energy storage organ, but also a secretary organ, producing a variety of bioactive substances, including adiponectin. Adiponectin is paradoxically reduced in obesity and elevated adiponectin concentration is associated with greater insulin sensitivity. Therefore hypoadiponectinemia can be considered a key factor of the development of METS. We believe that detection, prevention and treatment of METS are important clinical and public health challenges.

Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana.

We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity). Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups. The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases. Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs. The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots. We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.

Analysis of Tag1-like elements in Arabidopsis thaliana and their distribution in other plants.

Analysis of genomic DNA of Arabidopsis Columbia (Col.) ecotype using a transposon Tag1-specific primer showed the presence of Tag1 homologues which was confirmed by Southern hybridization with a Tag1 probe. Further analysis showed that the homologue, 0.75 kb in length, had inverted repeats at both ends, 8-bp duplicated sequences at the site at which it is located and about 80% homology with Tag1, and was randomly distributed in the Arabidopsis genome. Based on these results, we concluded that these elements are non-autonomous variants of Tag1 and we termed this element sTag1. Using the polymerase chain reaction fragment hybridization technique, we found the distribution of such homologues in other plant species.

Cloning and characterization of a plant gene encoding a protein kinase.

The cloning and sequence analysis of a gene that encodes a homologue of protein kinase (PK) from Arabidopsis thaliana is reported. We screened a genomic DNA library of A. thaliana using as probes oligodeoxyribonucleotides or fragments from the polymerase chain reaction that correspond to conserved regions in the catalytic domains of various PKs. One genomic clone, named Atpk5, was sequenced and analyzed. Transcripts of the corresponding gene, Atpk5, were detected in root, leaf and flower tissues by Northern blot analysis. The deduced amino acid sequence of the putative product of Atpk5 resembles those of kinases that phosphorylate ribosomal protein S6, cAMP-dependent PKs and protein kinase C. From the results of sequence comparisons, the ATPK5 protein appears to be a member of a subfamily of Ser/Thr-PKs specific to plants.

Construction of a cDNA library for a specific region of a chromosome using a novel cDNA selection method utilizing latex particles.

A novel method is described for the rapid concentration of particular cDNAs and their mapping to specific regions of a genome. The strategy for 'cDNA scanning' is based on the hybridization of an entire library of cDNAs to a large fragment of genomic DNA that is covalently bound to latex particles. The hybridized cDNAs are eluted, amplified by PCR and cloned into a lambda vector. Selected cDNAs that hybridized to the genomic DNA are cloned, with subsequent sequence analysis. Region-specific DNA fragments prepared from a yeast artificial chromosome (YAC) clone, EG10D9, which maps to chromosome 5 of the small cruciferous plant Arabidopsis thaliana (At), were used to prepare a model system and were covalently bound to latex particles. cDNAs that hybridized to EG10D9 were concentrated by hybridization to the immobilized DNA. The hybridized cDNAs were recovered and amplified by PCR. The resultant sub-library of cDNAs of 0.5-2 kb in length was enriched about 1000-fold. The partial sequences of the cDNAs provided information about genes that are located on the EG10D9 region of the At genome. The cDNA scanning strategy provides an efficient method for the mapping of expressed genes which could be used as expressed sequence tags (EST) within a genome.

The gene encoding a calcium-dependent protein kinase located near the sbe1 gene encoding starch branching enzyme I is specifically expressed in developing rice seeds.

A gene (spk) encoding a Ca(2+)-dependent protein kinase (SPK) is located in the region immediately upstream of the sbe1 gene encoding a starch branching enzyme. The spk gene is specifically expressed in developing seeds and its expression pattern is very similar to those of genes encoding starch-synthesizing enzymes such as sbe1 and waxy, seed lipid-synthesizing enzymes, as well as genes encoding seed storage proteins. A full-length spk cDNA was isolated from a cDNA library constructed from developing seeds. The deduced amino acid sequence showed that SPK has a high degree of homology to soybean and carrot Ca(2+)-dependent protein kinase, both of which contain calmodulin domains. The calmodulin domain, as well as the catalytic subdomain consensus regions of protein kinases are highly conserved in SPK. These results suggest that a tissue- and stage-specific protein kinase, SPK, is involved in the synthesis of seed storage compounds during seed development. They also strongly suggest that Ca2+ is required for seed development.

Characterization of a gene that encodes a homologue of protein kinase in Arabidopsis thaliana.

Cloning and analysis of a gene that encodes a homologue of protein kinase (PK) from Arabidopsis thaliana is reported. Oligodeoxyribonucleotides (oligos) corresponding to conserved regions in catalytic domains of various PKs were used for polymerase chain reaction (PCR) amplification with genomic DNA from A. thaliana as template, in an attempt to identify genes encoding PK in plants. We obtained several amplified DNA fragments that encoded part of a PK. We screened a genomic DNA library of A. thaliana with these oligos or PCR fragments as probes. Three genomic clones were obtained and one of them, named Atpk7, was sequenced and analyzed. Atpk7 was demonstrated by PCR to contain an intron. The mRNA transcribed from Atpk7 was detected predominantly in root tissue by Northern blot analysis. The transcription start point was determined by primer extension. The deduced amino acid (aa) sequence of the putative product of Atpk7 resembles those of S6 kinases, cyclic nucleotide-dependent PKs and calcium-dependent PKs. From this comparison of aa sequences, the ATPK7 protein is considered to be a member of a novel subfamily of Ser/Thr PKs in plants.

Establishment of framework P1 clones for map-based cloning and genome sequencing: direct RFLP mapping of large clones.

Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking. The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome. Using a P1-based library of Arabidopsis thaliana with genomic inserts of 70-90kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittier, R.F., 1995. Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking. Plant J. 7, 351-358), we have now established such a set of framework clones. To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs). In order to establish framework clones more efficiently, we refined protocols for P1 clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes. This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes. Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap.

Two genes that encode ribosomal-protein S6 kinase homologs are induced by cold or salinity stress in Arabidopsis thaliana.

We have isolated two closely related cDNA clones (cATPK19 and cATPK6) with homology to protein-serine/threonine kinases from Arabidopsis thaliana using the polymerase chain reaction (PCR). The deduced amino acid sequences of the ATPK19 and ATPK6 contain all 11 conserved regions of the catalytic domain of protein kinases, and have homology to p70 ribosomal S6 kinases (52%). ATPK19 and ATPK6 have putative PEST regions in their N- and C-terminal regions, and these regions also contain putative phosphorylation sites that are recognized by casein kinases or proline-directed protein kinases such as cdc2, MAP kinase, and p54 MAP-2 kinase (SAPK). The transcription levels of the ATPK19 and ATPK6 genes rapidly and markedly increased when plants were subjected to cold or high-salt stresses. These observations suggest that ATPK19 and ATPK6 may function in the adaptation of plant cells to cold or high-salt conditions, providing an understanding of the role of protein phosphorylation in plant responses to environmental stresses.

ATMPKs: a gene family of plant MAP kinases in Arabidopsis thaliana.

We previously reported two cDNAs for MAP kinases (cATMPK1 and cATMPK2) from a dicot plant, Arabidopsis thaliana. We describe here the cloning and characterization of five additional cDNAs encoding novel MAP kinases in Arabidopsis, cATMPK3, cATMPK4, cATMPK5, cATMPK6, and cATMPK7. The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of animal MAP kinases are conserved in all the seven putative ATMPK proteins. Genes for MAP kinases in Arabidopsis constitute a family that contains more than seven members. Sequence analysis suggests that there are at least three subfamilies in the family of Arabidopsis genes for MAP kinases.

Chloroplast ribosomal protein L32 is encoded in the chloroplast genome.

The 50 S subunit of chloroplast ribosomes was prepared from tobacco leaves. The proteins were fractionated and the N-terminal amino acid sequence of a 14 kDa protein was determined. This sequence matches the N-terminal sequence deduced from ORF55 located between ndhF and trnL on the small single-copy region of tobacco chloroplast DNA. The deduced protein shows homology to E. coli and B. stearothermophilus L32 proteins, and it has been named as CL32 and ORF55 as rpl32. The tobacco chloroplast genome therefore contains 21 different ribosomal protein genes.

Expression of 11beta-hydroxysteroid dehydrogenases in bovine follicle and corpus luteum.

In glucocorticoid target organs, local concentrations of active glucocorticoid are determined by the relative expression of two 11beta-hydroxysteroid dehydrogenases (HSDs): bi-directional 11beta-HSD type1 (11HSD1) that mainly activates cortisone to cortisol, and dehydrogenase 11beta-HSD type2 (11HSD2) that inactivates cortisol to cortisone. In this study, we examined the expression of mRNA encoding these two 11beta-HSDs in bovine granulosa cells harvested from preovulatory follicles and corpora lutea (CL). Ovaries were obtained from Holstein cows at a local slaughterhouse. Follicles larger than 10 mm in diameter and CL were dissected and follicular fluid and granulosa cells were taken. Corpora lutea were weighed and their stages were morphologically assessed (stage I, days 1-4; stage II, days 5-10; stage III, days 11-17; stage IV, days 8-20). Follicles were classified into four groups according to their hormonal status (oestradiol (E(2)): progesterone (P(4))>1: oestrogen active; E(2):P(4)<1: oestrogen inactive) and stage of the oestrous cycle (luteal or follicular phase). Total RNA was extracted with phenol-chloroform and subjected to a semi-quantitative RT-PCR for 11HSD1, 11HSD2 and beta-actin. Concentrations of steroids in follicular fluid were determined by an enzyme immunoassay. In granulosa cells, only 11HSD1 mRNA was detected. There was a negative correlation between the expression of 11HSD1 and the concentration of cortisol in follicular fluid (P<0.05), indicating 11HSD1 may act as a dehydrogenase in the bovine follicle. Both types of 11beta-HSDs were expressed in CL. The levels of mRNA for both isozymes were high in stage I and II, and were decreased in stage III CL. In stage IV CL, the expression of 11HSD2 but not 11HSD1 mRNA increased. These results indicate that the bovine granulosa cells and CL express 11HSD1 and 11HSD2, and they may play an important physiological role in the bovine ovary through modulating the local glucocorticoid environment.

Obstruction of St Jude Medical valves in the aortic position: significance of a combination of cineradiography and echocardiography.

BACKGROUND: Obstruction of the St Jude Medical valve (St Jude Medical, Inc, St Paul, Minn) is a rare but serious complication. METHODS: Cineradiographic and echocardiographic evaluations of aortic St Jude Medical valves were simultaneously performed on 54 patients, with no signs of prosthetic valve dysfunction late after surgery. RESULTS: Although closing angles of the leaflets corresponded closely with the manufacturer data, restricted opening of the leaflets (opening angle >/= 20 degrees ) was found in 16 (group D) of the 54 patients by means of cineradiography. The opening angles were equal to or less than 14 degrees in the other 23 patients (group N) and between 15 degrees and 19 degrees in the remaining 15 (group M). Doppler-derived transprosthetic pressure gradients were significantly higher (P =.03) and the velocity index was significantly lower (P =.003) in group D than in group N. However, no significant differences were found in those values between group N and group M. Replacement of the aortic St Jude Medical valves was performed in 5 of the 16 patients, and the remaining 11 have been followed up because of relatively low pressure gradients. The cause of restricted leaflet movement was pannus formation without thrombosis in 4 patients and valve thrombosis with pannus formation in one. CONCLUSIONS: Reduced valve orifice area and restricted opening of the leaflets resulting from excess growth of pannus probably led to obstruction of the aortic St Jude Medical valves. A combination of cineradiography and echocardiography makes it possible to provide an accurate and detailed diagnosis of obstruction of the valve.

Clinical evaluation with exercise performance in twenty patients who underwent coronary artery bypass grafting with both the gastroepiploic and internal thoracic arteries.

Postoperative exercise performance was evaluated in 20 patients who underwent complete coronary revascularization with simultaneous right gastroepiploic artery and internal thoracic artery grafts for ischemic heart disease and exhibited patency of all grafts on postoperative angiograms. Three patients received only arterial grafts, and 17 simultaneously received a saphenous vein graft. Forty five right gastroepiploic artery grafts were harvested during this study, but two were not used because of foci of severe calcification. The right gastroepiploic artery was grafted to the distal right coronary artery in 17 patients and to the distal obtuse marginal branches in three patients, accompanied by an internal thoracic artery graft to the left anterior descending artery in seventeen, to the diagonal branch in three, and to the proximal right coronary artery in one patient. A postoperative exercise test with a bicycle ergonometer was administered 6 to 12 weeks after the operation, and the results are reported after conversion into metabolic units. The preoperative exercise test resulted in 3.6 +/- 1.9 metabolic units for 18 patients tested, and the postoperative metabolic units were 7.8 +/- 1.3 for 20 patients. Left ventricular wall motion was analyzed by regional ejection fraction before and after coronary artery bypass grafting in 14 patients who received an right gastroepiploic artery graft for the right coronary system. The mean regional ejection fraction of the posterobasal or diaphragmatic wall was 28.0% or 28.2% before operation and improved to 43.1% or 43.2% after coronary artery bypass graft, respectively. The flow of right gastroepiploic artery seemed to distribute adequately to the revascularized area. Long-term results have not yet been proved; the use of simultaneous right gastroepiploic artery and internal thoracic artery grafts, however, thus produced excellent results, as seen from improvements in both left ventricular wall motion and early postoperative exercise performance.

Augmentation of interleukin-2 production after cardiac operations in patients treated with erythropoietin.

It has been reported recently that cardiac operations can be followed by a transient impairment of cell-mediated immunity. In this study we examined indices of cell-mediated immunity in patients undergoing cardiac operations. Twenty-five of the patients received erythropoietin (200 U/kg) daily for 2 weeks before and after operation, and 30 matched control patients did not receive erythropoietin. On postoperative day 1, the numbers of total T cells and helper/inducer T cells were significantly higher in erythropoietin-treated patients than in control patients. The ability of patients' cells to make interleukin-2 in vitro increased after erythropoietin injection in the preoperative period. By postoperative day 1, erythropoietin-treated patients exhibited a fall in interleukin-2 production that was significantly less than that in control patients; levels increased by day 2 to a mean value twice that of the preoperative baseline and more than four times the corresponding mean level in the control groups, and levels returned to the baseline range by postoperative day 14. Levels of interleukin-2 production in erythropoietin-treated patients were significantly higher than those in control patients at each interval tested through postoperative day 7. These findings indicate that erythropoietin treatment not only augmented levels of circulating erythrocytes but also improved indices of cell-mediated immunity. Although the mechanism responsible for this effect remains to be determined, the finding suggests that erythropoietin might help to ameliorate or prevent the impairment of immune function that can occur after cardiac operations.

Postoperative erythroderma after cardiac operations. The possible role of depressed cell-mediated immunity.

Erythroderma as a manifestation of graft-versus-host disease after cardiac operations with blood transfusion may occur more frequently in Japan than in other countries. We have seen this problem in five patients who, after heart operations, died with symptoms and signs characteristic of graft-versus-host disease: cutaneous eruption, fever, diarrhea, leukopenia associated with agranulocytosis, and liver dysfunction. In the three patients seen most recently, skin biopsy showed findings similar to those of graft-versus-host disease after bone marrow transplantation. In addition, immunologic investigation showed remarkable differences in the findings in these patients and in those who did not have a graft-versus-host disease-like syndrome after cardiac operations. In particular, interleukin-2 production in response to mitogen stimulation was markedly diminished after operation in our patients, and the ratio of OKT4+ cells to OKT8+ cells in peripheral blood was low, reflecting increased numbers of OKT8+ cells after the occurrence of symptoms. The results raise the possibility that transient depression of cellular immunity after cardiac operations with blood transfusion may contribute to the occurrence of postoperative acute graft-versus-host disease.

Effects of minimal-dose aprotinin on coronary artery bypass grafting.

OBJECTIVE: To evaluate the effects of minimal-dose aprotinin in patients undergoing coronary artery bypass grafting, we conducted a prospective randomized study. METHODS: A total of 167 patients were randomized to receive no aprotinin treatment (control, n = 57), minimal-dose aprotinin (1.0 x 10(6) KIU; n = 55), or low-dose aprotinin (2.7 +/- 0.5 x 10(6) KIU; n = 55). Blood loss and transfusion requirements, parameters of clotting and fibrinolysis, renal function, and early graft patency rates were assessed. RESULTS: Postoperative blood loss and transfusion requirements were significantly (p = 0.01) lower in both the minimal-dose and low-dose groups than in the control group. The increase in D-dimer level after cardiopulmonary bypass was significantly (p < 0.05) less marked in the low-dose group than in the control group. The alpha 2-plasmin inhibitor and plasminogen activator inhibitor-1 levels were significantly (p < 0.05) greater in the minimal-dose and low-dose groups than in the control group after bypass, suggesting the prevention of fibrinolysis by both aprotinin doses. No statistically significant differences in postoperative renal function and early vein graft patency rates were noted (control group, 93.8%; minimal-dose group, 95.5%; low-dose group, 92.3%; p = 0.25). CONCLUSIONS: Aprotinin was not associated with a significant increase in the prevalence of renal dysfunction or early vein graft occlusion. Minimal-dose aprotinin inhibited enhanced fibrinolytic activity and reduced blood loss and transfusion requirements after bypass equivalently to low-dose aprotinin. The dose of 1 x 10(6) KIU added to the pump prime may be acceptably effective in reducing blood loss in patients undergoing primary coronary operations.