PP091. Prematurity and hypertensive disorders in pregnancy. A major public health problem.

INTRODUCTION: Around the world each year 10% to 15% of direct maternal deaths are associated with hypertensive disorders in pregnancy. Not only it can be devastating and life threatening for the mothers but also for the babies. Risks for the baby include poor growth and prematurity [1]. In low and middle income countries many public hospitals have limited access to neonatal intensive care, and so the mortality and morbidity is likely to be considerably higher than in settings where such facilities are available. Thus, the approach to the problem preeclampsia should include issues related to prematurity and its impact, since many of these babies develop definitive complications, constituting a major public health problem with social and economic repercussions. OBJECTIVES: To evaluate the prevalence of preterm births in a public hospital health of the city of São Paulo, checking its main causal conditions, focusing in particular on the importance of hypertensive disorders in its determinism. METHODS: This was a retrospective study of a consecutive series of preterm infants, defined as gestational age less than 37 weeks born at Maternity School Vila Nova Cachoeirinha, situated at the north of Sao Paulo City, in the period from 01/04 to 31/12/2011. The study population was classified according to three main groups of causal factors: spontaneous labor, premature rupture of membranes and elective preterm delivery. In this group was evaluated the specific participation of hypertensive disorders. RESULTS: The number of live births during the study period was 5302 babies. Among these 433 had gestational age less than 37 weeks, corresponding to a prematurity rate of 8.16%. Of all infants, 385 cases were included in the analysis. Regarding the causes of the onset of preterm labor found that 140 cases (36.4%) presented with spontaneous labor, 128 cases (33.2%) had premature rupture of membranes and 117 (30.4%) cases were born as a result of elective preterm delivery. In the latter group 88 cases (75.2%) had complications related to hypertensive disorders. Regarding the general population of premature infants, hypertension accounted for 22.8% of cases. CONCLUSION: We conclude that among the various obstetric problems, hypertensive disorders represent an important impact on preterm birth in our setting, considering that our institution is a reference to this type of care across the city of São Paulo. This knowledge is a fundamental tool to support the adoption of interventions that can detect groups at risk for hypertension in pregnancy, the promotion of follow-up to intercept severe cases and provide an efficient network of maternity care that may have neonatal intensive care units. This set of measures is essential to minimize the impact of this serious problem.

Clathrin is involved in organization of mitotic spindle and phragmoplast as well as in endocytosis in tobacco cell cultures.

We previously identified a 175 kDa polypeptide in Lilium longiflorum germinating pollen using a monoclonal antibody raised against myosin II heavy chain from Physarum polycephalum. In the present study, the equivalent polypeptide was also found in cultured tobacco BY-2 cells. Analysis of the amino acid sequences revealed that the 175 kDa polypeptide is clathrin heavy chain and not myosin heavy chain. After staining of BY-2 cells, punctate clathrin signals were distributed throughout the cytoplasm at interphase. During mitosis and cytokinesis, clathrin began to accumulate in the spindle and the phragmoplast and then was intensely concentrated in the cell plate. Expression of the C-terminal region of clathrin heavy chain, in which light chain binding and trimerization domains reside, induced the suppression of endocytosis and the formation of an aberrant spindle, phragmoplast, and cell plate, the likely cause of the observed multinucleate cells. These data strongly suggest that clathrin is intimately involved in the formation of the spindle and phragmoplast, as well as in endocytosis.

Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro.

In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts.

Possible involvement of energy metabolism in the change of cytoplasm organization induced by a protein phosphatase inhibitor, calyculin A, in root hair cells of Limnobium stoloniferum.

In root hair cells of Limnobium stoloniferum, transvacuolar strands disperse and cytoplasmic spherical bodies (CSBs) emerge upon treatment with a protein phosphatase inhibitor, calyculin A (CA), whose effects were previously shown to be canceled by simultaneous treatment of the cells with a nonselective protein kinase inhibitor, K-252a. CSB formation is also suppressed by latrunculin B (LB) or cytochalasin D, actin filament depolymerization drugs, or 2,3-butanedione monoxime, an inhibitor of myosin activity. To confirm the involvement of myosin activity in CSB formation induced by CA, we examined the effect of an inhibitor of energy metabolism, NaN3, on CSB formation in root hair cells pretreated simultaneously with CA and LB. In the presence of CA-LB, CSB formation was suppressed due to the depolymerization of actin filaments. When these drugs were removed, the actin filaments recovered and CSBs emerged even in the presence of K-252a. These results indicated that the phosphorylation level in the cells is elevated during the CA-LB treatment and that a phosphorylation level sufficient for the CSB formation was sustained even after CA removal. On the other hand, CSB formation after simultaneous treatment with CA and LB was significantly suppressed in the presence of NaN3. In such cells, actin filament bundles recovered, although their organization was random. The present and previous results suggested that myosin activity is necessary for CSB formation induced by CA, and that myosin regulated by phosphorylation-dephosphorylation is implicated in the organization of the actin cytoskeleton in root hair cells.

FTY720, a novel immunosuppressive agent, induces apoptosis in human glioma cells.

FTY720, a metabolite from Isaria sinclairii, has been developed to be a potent immunosuppressive drug with induction of apoptosis in T cells and several cell lines. We investigated whether FTY720 induces apoptosis in human glioma cell lines, since they are relatively resistant to multiple apoptotic stimuli. In human glioma cells including T98G, FTY720 induced apoptosiswith ED50 between 1 to 10 microg/ml, while etoposidedid not induce apoptosis at the same doses. Among the caspase family proteases, mainly caspase-6 was activated during the apoptosis by FTY720 but not etoposide. In addition, FTY720 caused tyrosine dephosphorylation of FAK and did not activate a FAK-PI3-kinase survival pathway. This was confirmed also by the observation that orthovanadate prevented FTY720-induced dephosphorylation of FAK and inhibited FTY720-induced cell death. We assumed that FTY720 induced FAK dephosphorylation and cut off the FAK-PI3-kinase pathway resulting in the induction of apoptosis via caspase-6 activation in these glioma cells.

Direct observation of processive movement by individual myosin V molecules.

Myosin V is an unconventional myosin thought to move processively along actin filaments. To have hard evidence for the high processivity, we sought to observe directly the movement by individual native chick brain myosin V (BMV) molecules with fluorescent calmodulin. Single BMV molecules did exhibit highly processive movement along actin filaments fixed to a coverslip. BMV continued to move up to the barbed end of its actin track, and did not readily detach from action. The barbed end, therefore, got brighter with time, because of a constant stream of BMV traffic. The maximum speed of the processive movement was 1 microm/s, and the maximum actin-activated ATPase rate was 2.4 s(-1). These values apparently imply that BMV travels a great distance, 400 nm, per an ATPase cycle.

Calcium-calmodulin suppresses the filamentous actin-binding activity of a 135-kilodalton actin-bundling protein isolated from lily pollen tubes.

We have isolated a 135-kD actin-bundling protein (P-135-ABP) from lily (Lilium longiflorum) pollen tubes and have shown that this protein is responsible for bundling actin filaments in lily pollen tubes (E. Yokota, K. Takahara, T. Shimmen [1998] Plant Physiol 116: 1421-1429). However, only a few thin actin-filament bundles are present in random orientation in the tip region of pollen tubes, where high concentrations of Ca(2+) have also been found. To elucidate the molecular mechanism for the temporal and spatial regulation of actin-filament organization in the tip region of pollen tubes, we explored the possible presence of factors modulating the filamentous actin (F-actin)-binding activity of P-135-ABP. The F-actin-binding activity of P-135-ABP in vitro was appreciably reduced by Ca(2+) and calmodulin (CaM), although neither Ca(2+) alone nor CaM in the presence of low concentrations of Ca(2+) affects the activity of P-135-ABP. A micromolar order of Ca(2+) and CaM were needed to induce the inhibition of the binding activity of P-135-ABP to F-actin. An antagonist for CaM, W-7, cancelled this inhibition. W-5 also alleviated the inhibition effect of Ca(2+)-CaM, however, more weakly than W-7. These results suggest the specific interaction of P-135-ABP with Ca(2+)-CaM. In the presence of both Ca(2+) and CaM, P-135-ABP organized F-actin into thin bundles, instead of the thick bundles observed in the absence of CaM. These results suggest that the inhibition of the P-135-ABP activity by Ca(2+)-CaM is an important regulatory mechanism for organizing actin filaments in the tip region of lily pollen tubes.

Regulation of cytoplasmic pH under extreme acid conditions in suspension cultured cells of Catharanthus roseus: a possible role of inorganic phosphate.

Changes in cytoplasmic pH of suspension-cultured cells of Catharanthus roseus under extreme acid conditions were measured with the pH-dependent fluorescence dye; 2',7'-bis-(2-carboxyethyl)-5 (and-6) carboxyfluorescein (-acetoxymethylester) (BCECF). When cells were treated with 1 mM HCl (pH 3 solution), the cytoplasmic pH first decreased then returned to the original level. Treatment with 10 mM HCl (pH 2 solution) acidified the cytoplasm to a greater extent, and the acidification continued at a constant level throughout the measurement. Treatment with a pH 2 solution resulted in a gradual decrease of the malate content, indicating the operation of biochemical pH regulation mechanism. The pH 2 treatment also caused a sudden decrease of the intracellular level of Pi. The cellular content of total phosphorus did not change during the acidification. The Pi was converted to the organic phosphate form. The ATP level was not increased by the pH 2 treatment, but slightly decreased. The role of Pi, which might be functioning as a regulatory factor of cytoplasmic pH, a non-competitive inhibitor of the H+-pumps of both the plasma membrane and tonoplast is discussed.

The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis.

In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells.

Biochemical and immunocytochemical characterization of two types of myosins in cultured tobacco bright yellow-2 cells

We have isolated a myosin (referred to as 170-kD myosin) from lily pollen tubes, which consists of 170-kD heavy chain and calmodulin (CaM) light chain and is responsible for cytoplasmic streaming. A 170-kD polypeptide that has similar antigenicity to the 170-kD myosin heavy chain of lily pollen tubes was also present in cultured tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells, and possessed the ability to interact with F-actin in an ATP-dependent manner. In addition to this myosin, we identified biochemically another kind of myosin in BY-2 cells. This myosin consisted of a CaM light chain and a 175-kD heavy chain with antigenicity different from the 170-kD myosin heavy chain. In the present study, we referred to this myosin as 175-kD myosin. This myosin was able to translocate rhodamine-phalloidin (RP)-labeled F-actin at an average velocity of about 9 &mgr;m/s in the motility assay in vitro. In contrast, the sliding velocity of RP-labeled F-actin translocated by fractions containing the 170-kD myosin was 3 to 4 &mgr;m/s. The velocity of cytoplasmic streaming in living BY-2 cells ranged from 2 to 9 &mgr;m/s. The motile activity of 175-kD myosin in vitro was inhibited by Ca(2+) at concentrations higher than 10(-6) M. Immunoblot analyses using an antiserum against the heavy chain of 170- or 175-kD myosin revealed that in tobacco plants, the 175-kD myosin was expressed in leaf, stem, and root, but not in germinating pollen, while 170-kD myosin was present in all of these plant parts and in germinating pollen. These results suggest that the two types of myosins, 170 and 175 kD, presumably participate in cytoplasmic streaming in BY-2 cells and other somatic cells of tobacco plants.

The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity.

A plant 135-kDa actin-bundling protein (P-135-ABP) isolated from pollen tubes of Lilium longiflorum (Thunb.) binds stoichiometrically to F-actin filaments and bundles them in vitro (E. Yokota et al., 1998, Plant Physiol. 116: 1421-1429). To further understand the mechanism of actin-filament bundle formation by P-135-ABP, the polarity of each F-actin filament in bundles was examined using myosin subfragment 1 (S-1). Dissociation of F-actin filaments from bundles organized by P-135-ABP was induced by S-1. However, F-actin filaments that remained in a bundle and decorated by S-1 showed uniform polarity. These results indicate that P-135-ABP arranges F-actin filaments into bundles with uniform polarity and consequently plays a key role in the orientation of cytoplasmic streaming in pollen tubes.

FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line.

Protein phosphorylation in a human glioblastoma cell line, T98G, was examined after exposure to oxidative stress in vitro. Hydrogen peroxide (1 mM) markedly induced tyrosine phosphorylation of focal adhesion kinase (FAK) and serine phosphorylation of Akt at 1 h after stimulation. Concommitantly, the association of FAK with phosphatidylinositide 3'-OH-kinase (PI 3-kinase) was also observed by the hydrogen peroxide stimulation. When T98G cells were incubated with wortmannin, a PI 3-kinase inhibitor, both PI 3-kinase activity and phosphorylation of Akt were inhibited, whereas apoptosis by oxidative stress was accelerated. Concomitant with apoptosis, elevated level of CPP32 protease activity (caspase-3) was observed, with decreases in Bcl-2 protein and increases in Bax protein. These results suggested that in the signal transduction pathway from FAK to PI 3-kinase, Akt promotes survival. Thus, it became apparent that FAK is the upstream signal protein of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis in T98G cells.

Genetic diversities of hantaviruses among rodents in Hokkaido, Japan and Far East Russia.

Seroepizootiologic surveys among wild rodents were carried out in Japan and Far East Russia in 1995 and 1996. Seropositive animals were only identified in Clethrionomys rufocanus (23/134) in Hokkaido, Japan. On the other hand, seropositives were identified in C. rufocanus (1/8), Apodemus agrarius (2/66), Apodemus spp. (2/26) and Microtus fortis (3/22) in Vladivostok, Far East Russia. Total RNA was isolated from lungs of seropositive animals and the S genome segments were amplified by PCR, cloned and sequenced. The S and M genomes of hantavirus, derived from Japanese C. rufocanus (Tobetsu genotype), were most closely related with Puumala viruses (76-79% nucleotide and 95% amino acid identities for S genome, 70-78% nucleotide and 87-92% amino acid identities for M genome). The recombinant nucleocapsid protein of Tobetsu genotype was antigenically quite similar with that of Sotkamo. These suggest that the virus endemic in Japanese C. rufocanus belongs to Puumala virus. Phylogenetic analysis indicates that the genotype forms a distinct lineage within Puumala viruses. Partial S segment (1-1251 nt), derived from seropositive M. fortis in Vladivostok, was sequenced and analyzed. The S genome segment, which was designated Vladivostok genotype, was most closely related with Khabarovsk virus (79% nucleotide and 90% amino acid identities) which was isolated from M. fortis.

Inhibitory regulation of higher-plant myosin by Ca2+ ions

Myosin isolated from the pollen tubes of lily (Lilium longiflorum) is composed of a 170-kD heavy chain (E. Yokota and T. Shimmen [1994] Protoplasma 177: 153-162). Both the motile activity in vitro and the F-actin-stimulated ATPase activity of this myosin were inhibited by Ca2+ at concentrations higher than 10(-6) M. In the Ca2+ range between 10(-6) and 10(-5) M, inhibition of the motile activity was reversible. In contrast, inhibition by more than 10(-5) M Ca2+ was not reversible upon Ca2+ removal. An 18-kD polypeptide that showed the same mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that of spinach calmodulin (CaM) was present in this myosin fraction. This polypeptide showed a mobility shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a Ca2+-dependent manner. Furthermore, this polypeptide was recognized by antiserum against spinach CaM. By immunoprecipitation using antiserum against the 170-kD heavy chain, the 18-kD polypeptide was coprecipitated with the 170-kD heavy chain, provided that the Ca2+ concentration was low, indicating that this 18-kD polypeptide is bound to the 170-kD myosin heavy chain. However, the 18-kD polypeptide was dissociated from the 170-kD heavy chain at high Ca2+ concentrations, which irreversibly inhibited the motile activity of this myosin. From these results, it is suggested that the 18-kD polypeptide, which is likely to be CaM, is associated with the 170-kD heavy chain as a light chain. It is also suggested that this polypeptide is involved in the regulation of this myosin by Ca2+. This is the first biochemical basis, to our knowledge, for Ca2+ regulation of cytoplasmic streaming in higher plants.

[Hemophagocytic syndrome in a patient with rheumatoid arthritis].

A 62-year-old man with 16 year-history of rheumatoid arthritis (RA) was admitted due to progressive pancytopenia, general fatigue, and high fever. He was treated with 5 mg methotrexate weekly in RA. His bone marrow examination revealed a decreased nuclear cell count (2.1 x 10(4)/microliter), megakaryocyte count (16/microliter), and macrophages phagocytizing blood cells (13.2%), indicating the presence of hemophagocytic syndrome. No infections agent was detected in cultures derived from his blood or other sources. The serological tests for several viruses revealed no obvious viral etiology. The systemic lymphonodes were not swelling. Administration of 40 mg prednisolone daily improved his abnormal hematological findings. This is a case of RA accompanied by hemophagocytic syndrome, which is a rare complication of RA.

[Acute mitral regurgitation caused by malignant lymphoma of the posterior mediastinum].

An 82-year-old man was admitted to our hospital in September 1996 due to dysphagia and cardiomegaly. Physical examination detected the fourth heart sound and a Levine III/VI systolic murmur in the cardiac apex. Surface lymph nodes were not palpable. LDH 662 IU/I was detected by laboratory examinations, and ultrasound cardiography showed grade 3 mitral regurgitation. Computed tomography revealed a huge mass in the posterior mediastinum, pressing the heart from the posterior direction. Thereafter, a left pleural effusion developed and aspiration was performed. Cytological examination of the fluid showed clusters of lymphoid cells with a positive immunophenotype for CD10, CD19 and HLA-DR. Chromosome analysis revealed complex abnormal karyotypes including t(8;14) (q24;32). A diagnosis of B cell lymphoma was made, and combination chemotherapy consisting of cyclophosphamide, THP-adriamycin, vincristine, and prednisolone was initiated. The patient's mass disappeared promptly, and his mitral reguration subsided. We reported this case because malignant lymphoma of the posterior mediastinum is rare, and because we are unaware of any previous reports of malignant lymphoma causing acute mitral regurgitation.

[A case of Sjögren's syndrome associated with advanced hemolytic anemia caused by worsening autoimmune cholangiopathy].

We report a rate case of autoimmune cholangiopathy (AIC) and autoimmune hemolytic anemia (AIHA) in a patient with Sjögren's syndrome. A 59-year-old woman was admitted to Matsuyama Red Cross Hospital in September 1996 because of worsening liver dysfunction. She had suffered from keratoconjunctivitis sicca and xerostomia and had been diagnosed as having Sjögren's syndrome in February 1994, based on histological examination of the minor salivary gland and sialography, and positivity for SS-A and SS-B antibody. Liver dysfunction had first become evident in September 1995. Histological examination of a liver biopsy specimen obtained by laparoscopy showed that the structure of the hepatic lobules was mostly preserved, whereas most of the biliary ducts were transformed, being consistent with AIC. On admission, the patient was given 40 mg of prednisolone. Although the serum transaminase level decreased, jaundice persisted and hemolytic anemia developed. Further administration of 60 mg of prednisolone and plasmapheresis ameliorated the hemolytic anemia and cured the jaundice. We consider that an increased immunological response caused by the worsening AIC might have played a role in the development of AIHA in the present case.

[A case of the active intestinal tuberculosis detected during the examination of the right pleural effusion].

A 76-year-old female was admitted to our hospital because of fever and the right pleural effusion. On the analysis of pleural effusion, the total cell count was 6720/microliter with 95% lymphocytes, and ADA was 38.1 U/l. The culture of pleural effusion was negative, and the smear and PCR for Mycobacterium were also negative. For examinations, we performed eterography that showed cicatricial strictures of intestine. X-ray examination of the colonated colonoscopy showed ulcers (circular type), shortening of the colon, Bauhin's value insufficiency and diverticulum-like deformity. Then, she was diagnosed as intestinal tuberculosis. The smear and PCR of biopsy specimens from the lesion were positive, and antituberculotic therapy was effective. Finally, the culture of pleural effusion for Mycobacterium tuberculosis was positive after 8 weeks. We thought intestinal examination may be useful for the diagnosis of tuberculosis, when lymphocyte-rich exudative pleural effusion of unknown etiology is seen.

Purification of an actin-binding protein composed of 115-kDa polypeptide from pollen tubes of lily.

From lily pollen tubes, an actin-binding protein composed of 115-kDa polypeptide was purified sequentially by co-precipitation method with F-actin, hydroxylapatite column, gel filtration column and DE-52 ion exchange column chromatography. This component displayed a tendency to aggregate in solutions of low ionic strength, indicating a hydrophilic characteristic. Under physiological ionic conditions, this component bound to F-actin in an actin-concentration-dependent was saturable manner. Binding of this component to F-actin was independent of ATP and Ca(2+)-concentrations. Fluorescent microscopy revealed that F-actin labeled with rhodamine-phalloidin showed bundling in the presence of this component. Judging from the lack of antibody cross-reactivity, this component does not seem to be related to alpha-actinin of skeletal muscle and plant 135-kDa actin-bundling protein. Therefore, this component is the F-actin binding protein, which has not been identified thus far in plant cells.