Unusual diagnosis of feline cardiac lymphoma using cardiac needle biopsy.

BACKGROUND: Cardiac tumors in cats are relatively rare, with lymphoma accounting for more than half of all cases. However, feline cardiac lymphoma is often diagnosed post-mortem, and it is difficult to diagnose while the cat is still alive. It is the first report of a direct, rather than estimative, diagnosis with cardiac needle biopsy of a living cat with cardiac lymphoma. CASE PRESENTATION: A 3-year-old domestic short-haired male cat experienced loss of energy and loss of appetite. Thoracic radiography and transthoracic echocardiography showed cardiomegaly with slight pleural effusion and cardiac tamponade due to pericardial effusion, respectively. In addition, partial hyperechoic and hypertrophy of the papillary muscle and myocardium were observed. Blood test showed an increase in cardiac troponin I levels. Pericardial fluid, removed by pericardiocentesis, was analyzed; however, the cause could not be determined. With the owner's consent, pericardiectomy performed under thoracotomy revealed a discolored myocardium. Cardiac needle biopsy was performed with a 25G needle, and a large number of large atypical lymphocytes were collected; therefore, a direct diagnosis of cardiac lymphoma was made. Pathological examination of the pericardium diagnosed at a later date revealed T-cell large cell lymphoma. The cat underwent chemotherapy followed by temporary remission but died 60 days after the diagnosis. Postmortem, two-dimensional speckle-tracking echocardiography (data when alive) revealed an abnormal left ventricular myocardial deformation, which corresponded to the site of cardiac needle biopsy. CONCLUSIONS: This rare case demonstrates that cardiac lymphoma should be added to the differential diagnosis in cats with myocardial hypertrophy and that the diagnosis can be made directly by thoracotomy and cardiac needle biopsy. In addition, the measurement of cardiac troponin I levels and local deformation analysis of the myocardium by two-dimensional speckle-tracking echocardiography may be useful in the diagnosis of cardiac tumors.

Increased undercarboxylated osteocalcin/intact osteocalcin ratio in patients undergoing hemodialysis.

UNLABELLED: Serum undercarboxylated osteocalcin (ucOC)/intact osteocalcin (iOC) ratio increased >1.0 in the patients undergoing hemodialysis, particularly in those with high bone turnover state. Consequently, serum ucOC/iOC ratio might lose its significance as a bone metabolic marker to indicate vitamin K deficiency in hemodialysis patients. INTRODUCTION: Serum intact osteocalcin (iOC), undercarboxylated OC (ucOC), and the ucOC/iOC ratio are considered clinically relevant indices in pre-dialysis chronic kidney disease (CKD) and hemodialysis (HD) patients, despite their accumulation in uremic serum. METHODS: Serum iOC and ucOC were measured along with serum intact parathyroid hormone (iPTH), bone alkaline phosphatase (BAP), and tartrate-resistant acid phosphatase (TRACP)-5b in 89 pre-dialysis CKD and 189 HD patients. RESULTS: Serum iOC and ucOC showed significantly negative correlations with estimated glomerular filtration rate in pre-dialysis CKD patients, although serum ucOC/iOC ratio did not correlate. Serum ucOC was significantly greater in HD patients than in pre-dialysis CKD patients, while serum iOC did not differ significantly, resulting in serum ucOC/iOC ratio >1.0 in 135 (71.4%) out of 189 HD patients. HD patients with high serum ucOC/iOC ratio (>1.0) had a significantly younger age and significantly higher values of body mass index, serum creatinine, albumin, phosphate, iPTH, and TRACP-5b than those with low ucOC/iOC ratio (≤ 1.0). The baseline iPTH and P1NP correlated with the changes of the ucOC/iOC ratio during the 2 days of the inter-dialytic period. Multivariate analysis showed that log [ucOC/iOC] in HD patients was significantly associated with log [iPTH], log [BAP], or log [TRACP-5b]. CONCLUSIONS: Serum ucOC/iOC ratio >1.0 was observed in as high as 71.4% of HD patients, preferentially with high bone turnover state, in comparison with pre-dialysis CKD patients. These data suggested that serum ucOC/iOC ratio might lose its significance as a bone metabolic marker to indicate vitamin K deficiency in HD patients.

Role of accumulated calcium in alleviating aluminum injury in wheat plants.

Aluminum (Al) sensitive wheat cultivar kalyansona was grown for 14 d in a range of Ca solution (125, 625, and 2500 µM) plus other nutrients without Al. At 14 d after Ca treatment, half of these plants were harvested (H1), and the rest of the plants were exposed to 100 µM Al for additional 6 d and harvested (H2). Severe Al injury was found only in the plants with the lowest supply of Ca before Al treatment. Aluminum concentration in the apoplastic fluid was very high at 125 µM Ca probably because the plasma membrane of some of the cells was destroyed due to the attack of 100 µM Al. Aluminum content in roots decreased with increasing supply of Ca before Al treatment. Calcium content decreased drastically at harvest (H2) in the plants with 100 µM Al. Under Al stress conditions, the plant responded to Al in different ways due to not only the different Ca supply but also the variation of Ca content in the plant tissues. Actually, the plants having the largest Ca content in the roots before Al treatment can receive less Al injury during Al treatment. To substantiate this idea, a companion study was conducted to investigate the effects of 2500 µM Ca supply during, before, and after 100 µM Al treatment on root growth. The results indicated clearly that exogenous Ca supply before Al treatment is able to alleviate Al injury but less effective than Ca supply during Al treatment.

Short communication: effects of serum obtained from dairy cows with low or high body condition score on in vitro embryo development.

The objective of the study was to determine whether the serum obtained from animals differing in body condition score (BCS) affects in vitro embryo development. After in vitro fertilization, serum obtained from dairy cows of either low (L-BCS; 2.1 ± 0.14 on a scale of 1 to 5) or high BCS (H-BCS; 4.0 ± 0.0), or commercially available bovine serum (control) was added at 5% concentration to the in vitro culture medium. Use of serum obtained from H-BCS cows increased the cleavage rates compared with control serum at both 24 and 48 h after in vitro fertilization (78.3 vs. 71.9% and 79.9 vs. 75.1%, respectively), whereas use of serum obtained from L-BCS cows increased the blastocyst rate compared with control serum at 7d (23.8 vs. 19.1%), but this difference was not evident at 8 or 9 d after in vitro fertilization. As nonesterified fatty acid concentrations were highest in control serum, followed by serum from L-BCS and H-BCS cows (621, 559, and 272 µEq/L, respectively), a high concentration of nonesterified fatty acids might adversely affect the very early stages of embryo development, and its negative effects might be greater immediately after fertilization compared with developmental stages after morula formation. Our findings also indicate that factors promoting early stage embryo development do not necessarily promote blastocyst development. Serum obtained from animals under different physiological conditions may be used for in vitro embryo culture to study the effects of nutritional management of dairy cattle on embryo development.

Impact of polyplex micelles installed with cyclic RGD peptide as ligand on gene delivery to vascular lesions.

Gene therapy is expected to open a new strategy for the treatment of refractory vascular diseases, so the development of appropriate gene vectors for vascular lesions is needed. To realize this requirement with a non-viral approach, cyclo(RGDfK) peptide (cRGD) was introduced to block copolymer, poly(ethylene glycol)-block-polycation carrying ethylenediamine units (PEG-PAsp(DET)). cRGD recognizes α(v)ß(3) and α(v)ß(5) integrins, which are abundantly expressed in vascular lesions. cRGD-conjugated PEG-PAsp(DET) (cRGD-PEG-PAsp(DET)) formed polyplex micelles through complexation with plasmid DNA (pDNA) and the cRGD-PEG-PAsp(DET) micelles achieved significantly more efficient gene expression and cellular uptake as compared with PEG-PAsp(DET) micelles in endothelial cells and vascular smooth muscle cells. Intracellular tracking of pDNA showed that cRGD-PEG-PAsp(DET) micelles were internalized via caveolae-mediated endocytosis, which is associated with a pathway avoiding lysosomal degradation and that, PEG-PAsp(DET) micelles were transported to acidic endosomes and lysosomes via clathrin-mediated endocytosis. Further, in vivo evaluation in rat carotid artery with a neointimal lesion revealed that cRGD-PEG-PAsp(DET) micelles realized sustained gene expression, whereas PEG-PAsp(DET) micelles facilitated rapid, but transient gene expression. These findings suggest that introduction of cRGD to polyplex micelles might create novel and useful functions for gene transfer and contribute to the establishment of efficient gene therapy for vascular diseases.

Effects of folic acid on the development and oxidative stress of mouse embryos exposed to heat stress.

The development of mammalian pre-implantation embryos is inhibited by heat stress, and the inhibitory effect is associated with excess reactive oxygen species (ROS). Folate is a nutrient with various physiological functions including antioxidative effects. We first investigated the transcript expression for 10 enzymes in the cycle of folate metabolism (folate-methionine cycle) in mouse embryos at the 1-cell, 2-cell, 4- to 8-cell, morula and blastocyst stages using reverse transcription-polymerase chain reaction. All of the transcripts were consistently expressed, except for Mat1a, which was not detected from the 4- to 8-cell stage onward. Next, the effects of folic acid (the synthetic form of folate) on the development and ROS levels of heat-stressed embryos were investigated. One-cell mouse embryos were cultured with or without 1000 ng/ml folic acid basically at 38°C, and in the heat-stressed groups, embryos were exposed to 39.5°C/10 h/day on the first two days of culture. The heat stress significantly (p < 0.05) decreased blastocyst development and cell number and increased ROS levels compared to those in the group not subjected to heat stress; however, among the heat-stressed groups, blastocyst development and cell number were increased and the ROS level was decreased by the addition of folic acid. These results indicate that the mRNA of folate-methionine cycle enzymes are expressed in mouse pre-implantation embryos, suggesting they can independently utilize folate, and the inhibitory effects of heat stress on the development of mouse pre-implantation embryos are ameliorated by folic acid. The ameliorating effects of folic acid may be partly due to its antioxidative property.

Serum CRP in patients with gout and effects of benzbromarone.

OBJECTIVE: C-reactive protein (CRP) is associated with increased risk for myocardial infarction, atherosclerosis, and peripheral artery diseases, while increased serum uric acid level is suggested to be independently associated with an increased risk of cardiovascular mortality. Accordingly, to investigate whether hyperuricemia is associated with serum CRP, we compared serum CRP levels between healthy subjects and patients with gout. In addition, we also examined whether benzbromarone has effects on serum CRP levels in patients with gout and the expression of CRP messenger RNA of CRP in the hepatoma cell line HuH7. METHODS: In the first experiment, 40 healthy males and 43 male patients with gout were enrolled, then blood samples were drawn from each after an overnight fast. In the second experiment, 42 male patients with gout were given uric acid-lowering therapy with benzbromarone. Blood samples were drawn after an overnight fast before and 1 year after beginning benzbromarone treatment. In the third experiment, the effects of benzbromarone on IL1beta-induced CRP expression were determined in HuH7 cells. RESULTS: Log serum CRP levels were not significantly different between the patients with gout and healthy subjects, while log serum CRP levels were decreased by 11% after benzbromarone treatment, as compared to the values before treatment (p < 0.01). In addition, log serum adiponectin levels were elevated by 2% after treatment (p < 0.01). Furthermore, our in vitro findings demonstrated that benzbromarone down-regulated IL1beta-stimulated CRP gene expression. CONCLUSIONS: These results suggest that hyperuricemia may not contribute to an increase in serum CRP level, while benzbromarone may have a favorable effect on CRP.

Effects of bovine milk ingestion on urinary excretion of oxypurinol and uric acid.

OBJECTIVE: Although allopurinol is a xanthine oxidase inhibitor, its overall effect may be due to the action of oxypurinol, a metabolite of allopurinol and another xanthine oxidase inhibitor, since the biological half-life of oxypurinol is longer than that of allopurinol. Oxypurinol shares a renal transport pathway with uric acid and ingestion of bovine milk increases the urinary excretion of uric acid. Therefore, we investigated whether its ingestion promotes the urinary excretion of oxypurinol. SUBJECTS/METHODS: Bovine milk (15 ml/kg body weight) was administered to 6 healthy subjects who took allopurinol (300 mg) 12 h prior to ingestion. In addition, a control experiment was performed with the same subjects using the same protocol, except for the ingestion of water instead of bovine milk. Blood and urine samples were collected before and after bovine and water ingestion. RESULTS: In the bovine milk ingestion experiment, the urinary excretion values of oxypurinol and uric acid were increased by 18% and 38%, respectively, and the fractional excretion values of oxypurinol and uric acid were increased by 20% and 40%, respectively, whereas those did not change in the control experiment. In addition, the concentration of alanine and sum of concentrations of amino acids were increased by 16% and 20%, respectively, in the bovine milk ingestion experiment. CONCLUSION: These results suggest that bovine milk ingestion promotes the urinary excretion of oxypurinol as well as uric acid by increasing amino acid concentration.

Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesions in human endothelial cell apoptosis.

Normal endothelial and epithelial cells undergo apoptosis when cell adhesion and spreading are prevented, implying a requirement for antiapoptotic signals from the extracellular matrix for cell survival. We investigated some of the molecular changes occurring in focal adhesions during growth factor deprivation-induced apoptosis in confluent monolayers of human umbilical vein endothelial cells. Among the first morphologic changes after initiation of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the matrix followed by detachment. We observe a specific proteolytic cleavage of focal adhesion kinase (pp125FAK), an important component of the focal adhesion complex, and identify pp125FAK as a novel substrate for caspase-3 and caspase-3-like apoptotic caspases. The initial cleavage precedes detachment, and coincides with loss of pp125FAK and paxillin from focal adhesion sites and their redistribution into the characteristic membrane blebs of apoptotically dying cells. Cleavage of pp125FAK differentially affects its association with signaling and cytoskeletal components of the focal adhesion complex; binding of paxillin, but not pp130(Cas) (Cas, Crk-associated substrate) and vinculin, to the COOH terminally truncated pp125FAK is abolished. Therefore, caspase-mediated cleavage of pp125FAK may be participating in the disassembly of the focal adhesion complex and actively interrupting survival signals from the extracellular matrix, thus propagating the cell death program.

Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice.

Four full-thickness skin wounds made in normal mice led to the significant increase in levels of nerve growth factor (NGF) in sera and in wounded skin tissues. Since sialoadenectomy before the wounds inhibited the rise in serum levels of NGF, the NGF may be released from the salivary gland into the blood stream after the wounds. In contrast, the fact that messenger RNA and protein of NGF were detected in newly formed epithelial cells at the edge of the wound and fibroblasts consistent with the granulation tissue produced in the wound space, suggests that NGF was also produced at the wounded skin site. Topical application of NGF into the wounds accelerated the rate of wound healing in normal mice and in healing-impaired diabetic KK/Ta mice. This clinical effect of NGF was evaluated by histological examination; the increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of extracellular matrix were observed. NGF also increased the breaking strength of healing linear wounds in normal and diabetic mice. These findings suggested that NGF immediately and constitutively released in response to cutaneous injury may contribute to wound healing through broader biological activities, and NGF improved the diabetic impaired response of wound healing.

Enhanced exudation of malate in the rhizosphere due to AtALMT1 overexpression in blackgram (Vigna mungo L.) confers increased aluminium tolerance.

Worldwide, 50% of soil is acidic, which induces aluminium (Al) toxicity in plants, as the phyto-availability of Al3+ increases in acidic soil. Plants responds to Al3+ toxicity by exuding organic acids into the rhizosphere. The organic acid responsible for Al3+ stress response varies from species to species, which in the case of blackgram (Vigna mungo L.) is citrate. In blackgram, an Arabidopsis malate transporter, AtALMT1, was overexpressed with the motive of inducing enhanced exudation of malate. Transgenics were generated using cotyledon node explants through Agrobacterium tumefaciens-mediated transformation. The putative transgenics were initially screened by AtALMT1-specific genomic DNA PCR, followed by quantitative PCR. Two independent transgenic events were identified and functionally characterized in the T3 generation. The transgenic lines, Line 1 and 2, showed better root growth, relative water content and chlorophyll content under Al3+ stress. Both lines also accounted for less oxidative damage, due to reduced accumulation of ROS molecules. Photosynthetic efficiency, as measured in terms of Fv /Fm , NPQ and Y(II), increased when compared to the wild type (WT). Relative expression of genes (VmSTOP1, VmALS3, VmMATE) responsible for Al3+ stress response in blackgram showed that overexpression of a malate transporter did not have any effect on their expression. Malate exudation increased whereas citrate exudation did not show any divergence from the WT. A pot stress assay found that the transgenics showed better adaptation to acidic soil. This report demonstrates that the overexpression of a malate transporter in a non-malate exuding species improves adaptation to Al3+ toxicity in acidic soil without effecting its stress response mechanism.

Uracil-tegafur and tamoxifen vs cyclophosphamide, methotrexate, fluorouracil, and tamoxifen in post-operative adjuvant therapy for stage I, II, or IIIA lymph node-positive breast cancer: a comparative study.

BACKGROUND: It has been reported that treatment with uracil-tegafur (UFT) has shown significantly better survival and relapse-free survival (RFS) than surgery alone. Therefore, we compared UFT with a combination therapy of cyclophosphamide, methotrexate, and fluorouracil (CMF) in patients who had undergone curative surgery for axillary lymph node-positive breast cancer. METHODS: A total of 377 node-positive patients with stage I, II, or IIIA disease were registered from September 1996 through July 2000 and were randomly assigned to either 6 cycles of CMF or 2 years of UFT. In both arms, tamoxifen (TAM) was concurrently administered for 2 years. The primary end point in this study was the non-inferiority of UFT to CMF. RESULTS: No statistically significant difference between the two groups was observed with regard to the 5-year RFS rate (72.2% in the UFT and 76.3% in the CMF). Adverse event profiles differed between the two groups, with a significantly lower incidence of leukopenia and anaemia in the UFT group, as well as anorexia, nausea/vomiting, stomatitis, and alopecia, which have implications for quality of life. CONCLUSION: UFT administered in combination with TAM holds promise in the treatment of lymph node-positive early breast cancer. On stratified analysis, the recurrence rate in the UFT group was found to be better in oestrogen receptor (ER)-positive patients. Tegafur-based treatment should be evaluated by a prospective randomised trial conducted in ER-positive patients.

Isolation and structure determination of blepharismin, a conjugation initiating gamone in the ciliate blepharisma.

One of the gamones (gamone II) which are effective for the induction of conjugation in Blepharisma intermedium has been isolated in a crystalline form and designated as blepharismin. From the result of chemical and spectroscopic investigations, in which x-ray crystallographic analysis was used as a definitive tool, blepharismin has been found to have the structure of calcium 3-(2'-formylamino-5'-hydroxybenzoyl)lactate.

Selective and sustained delivery of basic fibroblast growth factor (bFGF) for treatment of peripheral arterial disease: results of a phase I trial.

OBJECTIVES: The aim of this study was to evaluate the safety of selective and sustained delivery of basic fibroblast growth factor (bFGF) using acidic gelatine hydrogel microspheres (AGHMs) for the treatment of peripheral arterial disease (PAD). MATERIALS AND METHODS: We conducted a non-randomised and uncontrolled trial involving prospective observation of eight patients (eight limbs) with PAD - five limbs with arteriosclerosis obliterans and three limbs with thromboangiitis obliterans, five limbs (three arms and two legs) with critical limb ischaemia (CLI) and three limbs with intermittent claudication (IC) - who were followed up for 6 months or more. AGHM suspension containing 100 microg bFGF was infused into the artery of the affected limb. Besides evaluation of safety and changes in symptoms, resting ankle-brachial pressure index measurement and transcutaneous PO(2) (tcPO(2)), angiography were conducted at baseline and then at various time points. Skin perfusion pressure as an index of CLI and claudication distance as an index of IC were also used to assess clinical improvement and limb perfusion. RESULTS: No serious adverse events were observed. All cases showed improvement in symptoms, although this was temporary in some patients. CONCLUSION: Selective delivery of bFGF using AGHMs was suggested to be safe and well-tolerated in patients with PAD.

Carrier and spin dynamics of high-density exciton magnetic polarons in Cd0.8Mn0.2Te.

We investigated the carrier and spin dynamics of high-density exciton magnetic polarons (HD-EMPs) in Cd0.8Mn0.2Te based on the measurement of their time-resolved photoluminescence (PL) spectra and polarization states, and the utilization of photo-induced Faraday rotation techniques. The PL from the HD-EMPs were collected in a forward scattering configuration, and was observed as a pulsed emission of a few picoseconds duration, exhibiting a blue-shift with time evolution. The blue shift originated from the refractive-index dispersion of the sample. By excluding the influence of the refractive-index dispersion on the time profile, it was revealed that the ultra-short pulsed emission with a time width smaller than 1 ps was initially radiated with a time delay of ~2.4 ps after photoexcitation. From the results of time evolution of the polarization states, it is concluded that the exciton-Mn spin interactions occurs immediately after the excitation, which causes the Mn ion spins to align to follow the spin states of photoexcited excitons. The alignment of the Mn ion spins through the formation of the HD-EMPs was significantly faster than that of the localized EMP. On the other hand, the time evolution of the photo-induced Faraday rotation showed two decay components attributed to spin relaxations of the excitons and Mn ions within the HD-EMP. The observation of the Faraday rotation signal due to the Mn ion spins further confirms that these spins were aligned by the photo-excited spin-aligned excitons. Our findings suggest a novel mechanism for the effective optical control of spins in a semimagnetic semiconductor, which is associated with a multi-exciton system and its localized state.

GhSTOP1, a C2H2 type zinc finger transcription factor is essential for aluminum and proton stress tolerance and lateral root initiation in cotton.

Aluminum (Al) and proton (H+ ) ions are major acid soil stress factors deleteriously affecting plant root growth and crop yield. In our preliminary studies, cotton (Gossypium hirsutum L.) seedlings displayed very sensitive phenotypes to Al and H+ rhizotoxicities. Based on previous Arabidopsis results, we aimed to characterise the Al stress responsive Sensitive to Proton rhizotoxicity 1 (GhSTOP1) transcription system in cotton using RNAi-mediated down-regulation. With the help of seed embryo apex explants, we developed transgenic cotton plants overexpressing a GhSTOP1-RNAi cassette with NPTII selection. Kanamycin-tolerant T1 seedlings were further considered for Al and H+ stress tolerance studies. Down-regulation of the GhSTOP1 increased sensitivity to Al and proton rhizotoxicities, and root growth was significantly reduced in RNAi lines. The expression profile of GhALMT1 (Aluminum-activated Malate Transporter 1), GhMATE (Multidrug and Toxic Compound Extrusion), GhALS3 (Aluminium Sensitive 3) and key genes involved in the GABA shunt were down-regulated in the transgenic RNAi lines. Additionally, the lateral root initiation process was delayed and expression of GhNAC1, which is involved in lateral roots, was also suppressed in transgenic lines. Besides, overexpression of GhSTOP1 in Arabidopsis accelerated root growth and AtMATE and AtALMT1 expression under Al stress conditions. These analyses indicate that GhSTOP1 is essential for the expression of several genes which are necessary for acid soil tolerance mechanisms and lateral root initiation.

The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle.

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.

Heterogeneity of beta-type myosin isozymes in the human heart and regulational mechanisms in their expression. Immunohistochemical study using monoclonal antibodies.

To investigate the existence of heterogeneity of beta-type myosin isozymes (HC beta) in human hearts, immunohistochemical studies using monoclonal antibodies (MoAbs) raised against human ventricular myosin heavy chains were performed. Two types of MoAbs recognized some muscle fibers in the atrium, whereas both reacted with all ventricular muscle fibers. Since atrial muscle fibers reactive with each MoAb were found to be clearly different, the existence of two immunologically distinct HC beta (beta 1, and beta 2) was suggested in the atrium. By using affinity chromatography, two molecular variants of HC beta were isolated from the bovine atrium, and differences in the primary structure of beta 1 and beta 2 were confirmed by analysis of peptides produced by chymotryptic digestion. In pressure-overloaded human atria, myofibers containing beta 1 and/or beta 2 increased in accordance with decrement of myofibers containing alpha-type myosin isozyme (P less than 0.01). But they differed in expression during the developmental stage, since beta 2 did not exist in the early embryonic bovine heart, but beta 1 did. Thus, there are two distinct HC beta whose expression is regulated by at least two factors: pressure overload and developmental stage.

The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets.

Activation of the classical mitogen-activated protein kinase (MAPK) pathway leads to proliferation of many cell types. Accordingly, an inhibitor of MAPK kinase, PD 098059, inhibits PDGF-induced proliferation of human arterial smooth muscle cells (SMCs) that do not secrete growth-inhibitory PGs such as PGE2. In striking contrast, in SMCs that express the inducible form of cyclooxygenase (COX-2), activation of MAPK serves as a negative regulator of proliferation. In these cells, PDGF-induced MAPK activation leads to cytosolic phospholipase A2 activation, PGE2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which acts as a strong inhibitor of SMC proliferation. Inhibition of either MAPK kinase signaling or of COX-2 in these cells releases them from the influence of the growth-inhibitory PGs and results in the subsequent cell cycle traverse and proliferation. Thus, the MAPK pathway mediates either proliferation or growth inhibition in human arterial SMCs depending on the availability of specific downstream enzyme targets.

Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells.

Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells.