C1-inhibitor autoantibodies in SLE.

The presence of anti-C1-inhibitor (anti-C1-INH) autoantibodies is a hallmark of acquired C1-inhibitor deficiency. However, only scarce data are available on their prevalence, diagnostic value, and/or significance in systemic lupus erythematosus (SLE). In a multicentre study, we determined the levels of autoantibodies to C1-inhibitor in sera from 202 patients with SLE and 134 healthy controls. Additional clinical and laboratory parameters, such as organ involvement, as well as anti-C1q, anti-double-stranded DNA antibody, erythrocyte sedimentation rate, C-reactive protein, C3 and C4 serum complement levels have been studied in patients. The level of anti-C1-INH IgG was significantly higher (p = 0.034) in SLE patients, than in the controls. A high anti-C1-INH level of > or =0.4 U/ml (mean of controls + 2 SD) was found in 17% of the patients, but in only 4% of the controls (p = 0.0003). The SLEDAI score was significantly higher (p = 0.048) and the duration of SLE was significantly longer (p = 0.0004) among patients with elevated anti-C1-INH levels compared with patients without this autoantibody (median disease duration 8 vs. 17 years, respectively). Anti-C1-INH level was not correlated with any other laboratory parameter or organ manifestation of the disease. These findings indicate that the anti-C1-INH level is higher in SLE patients than in healthy controls and furthermore, the anti-C1-INH level correlates with the duration and activity of the disease.

Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells.

A calmodulin like domain protein kinase (CPK) homologue was identified in alfalfa and termed MsCPK3. The full-length sequence of cDNA encoded a 535 amino acid polypeptide with a molecular weight of 60.2 kDa. The deduced amino acid sequence showed all the conserved motifs that define other members of this kinase family, such as serine-threonine kinase domain, a junction region and four potential Ca2+ -binding EF sites. The recombinant MsCPK3 protein purified from E. coli was activated by Ca2+ and inhibited by calmodulin antagonist (W-7) in in vitro phosphorylation assays. The expression of MsCPK3 gene increased in the early phase of the 2,4-D induced alfalfa somatic embryogenesis. Heat shock also activated this gene while kinetin, ABA and NaCl treatment did not result in MsCPK3 mRNA accumulation. The data presented suggest that the new alfalfa CPK differs in stress responses from the previously described homologues and in its potential involvement in hormone and stress-activated reprogramming of developmental pathways during somatic embryogenesis.

RNA helicase dynamics in pre-mRNA splicing.

The DExH-box NTPase/helicase Prp22p plays two important roles in pre-mRNA splicing. It promotes the second transesterification reaction and then catalyzes the ATP-dependent release of mature mRNA from the spliceosome. Evidence that helicase activity is important emerged from the analysis of Prp22p motif III (SAT) mutations that uncouple the NTPase and helicase activities. We find that S635A and T637A hydrolyse ATP, but are defective in unwinding duplex RNA and releasing mRNA from the spliceosome. The S635A mutation is lethal in vivo at

Multiple cyclin-dependent kinase complexes and phosphatases control G2/M progression in alfalfa cells.

Reversible phosphorylation of proteins by kinases and phosphatases plays a key regulatory role in several eukaryotic cellular functions including the control of the division cycle. Increasing numbers of sequence and biochemical data show the involvement of cyclin-dependent kinases (CDKs) and cyclins in regulation of the cell cycle progression in higher plants. The complexity represented by different types of CDKs and cyclins in a single species such as alfalfa, indicates that multicomponent regulatory pathways control G2/M transition. A set of cdc2-related genes (cdc2Ms A, B, D and F) was expressed in G2 and M cells. Phosphorylation assays also revealed that at least three kinase complexes (Cdc2Ms A/B, D and F) were successively active in G2/M cells after synchronization. Interaction between alfalfa mitotic cyclin (Medsa;CycB2;1) and a kinase partner has been reported previously. The present yeast two-hybrid analyses showed differential interaction between defined D-type cyclins and Cdc2Ms kinases functioning in G2/M phases. Localization of Cdc2Ms F kinase to the preprophase band (PPB), the perinuclear ring in early prophase, the mitotic spindle and the phragmoplast indicated a pivotal role for this kinase in mitotic plant cells. So far limited research efforts have been devoted to the functions of phosphatases in the control of plant cell division. A homologue of dual phosphatase, cdc25, has not been cloned yet from alfalfa; however tyrosine phosphorylation was indicated in the case of Cdc2Ms A kinase and the p(13suc1)-bound kinase activity was increased by treatment of this complex with recombinant Drosophila Cdc25. The potential role of serine/threonine phosphatases can be concluded from inhibitor studies based on okadaic acid or endothall. Endothall elevated the kinase activity of p(13suc1)-bound fractions in G2-phase alfalfa cells. These biochemical data are in accordance with observed cytological abnormalities. The present overview with selected original data outlines a conclusion that emphasizes the complexity of G2/M regulatory events in flowering plants.

Effect of 10%, 30%, and 60% body weight traction on the straight leg raise test of symptomatic patients with low back pain.

STUDY DESIGN: Single group test-retest repeated measures. OBJECTIVES: To determine the effects of lumbar traction with 3 different amounts of force (10%, 30% and 60% body weight) on pain-free mobility of the lower extremity as measured by the straight leg raise (SLR) test. BACKGROUND: There are several recommendations on how lumbar traction should be performed, but the duration, frequency, force, and type of technique to be applied differ among the sources. METHODS AND MEASURES: Ten subjects with subjective complaints of low back pain or radicular symptoms with a positive unilateral SLR test below 45 degrees participated in this study. The pain-free mobility of the lower extremity in the SLR test position was measured prior to and immediately following 5 minutes of static traction in the supine position. Random assignment in the order of the amount of applied traction was implemented. RESULTS: The straight leg raise measurements were found to be significantly greater immediately following 30% and 60% of body weight traction as compared to pretraction and 10% of body weight traction. The mean (SD) SLR measurements were pretraction (24.1 degrees +/- 13.0), 10% of body weight traction (27.4 degrees +/- 14.5), 30% of body weight traction (34.0 degrees +/- 14.3), 60% of body weight traction (36.5 degrees +/- 15.8). CONCLUSIONS: The results of this study indicate that traction in this group of patients improved the mobility of the lower extremity during the SLR test. Both 30% and 60% of body weight tractions were shown to be effective for increasing motion beyond pretraction levels.

Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa.

Reversible phosphorylation of serine/threonine residues of cell cycle-regulatory proteins is one of the key molecular mechanisms controlling eukaryotic cell division. In plants, the protein kinase partners (i.e. p34cdc2/CDC28-related kinases) have been extensively studied, while the role of counter-acting protein phosphatases is less well understood. We used endothall (ET) as a cell-permeable inhibitor of serine/threonine-specific protein phosphatases to alter cytological and biochemical characteristics of cell division in cultured alfalfa cells. A high concentration of ET (10 and 50 microM) inhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low concentration (1 microM) of ET-treatment primarily reduced the PP2A activity. High concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In contrast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exposure of cells to 1 microM ET resulted in disturbance of preprophase band formation, increase in the number of nuclei with prophase microtubule assembly, premature polarisation of the spindle, and abnormal phragmoplast maturation. Under the same conditions, the ET-treated cells exhibited an early increase in cdc2MsF kinase activity. These results suggest that PP2A contributes to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on both PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co-ordination of chromosomal and microtubule events in dividing plant cells.

Characterization of photosystem II activity and heterogeneity during the cell cycle of the green alga scenedesmus quadricauda

The photosynthetic activity of the green alga Scenedesmus quadricauda was investigated during synchronous growth in light/dark cycles. The rate of O2 evolution increased 2-fold during the first 3 to 4 h of the light period, remained high for the next 3 to 4 h, and then declined during the last half of the light period. During cell division, which occurred at the beginning of the dark period, the ability of the cells to evolve O2 was at a minimum. To determine if photosystem II (PSII) controls the photosynthetic capacity of the cells during the cell cycle we measured PSII activity and heterogeneity. Measurements of electron-transport activity revealed two populations of PSII, active centers that contribute to carbon reduction and inactive centers that do not. Measurements of PSII antenna sizes also revealed two populations, PSIIalpha and PSIIbeta, which differ from one another by their antenna size. During the early light period the photosynthetic capacity of the cells doubled, the O2-evolving capacity of PSII was nearly constant, the proportion of PSIIbeta centers decreased to nearly zero, and the proportion of inactive PSII centers remained constant. During the period of minimum photosynthetic activity 30% of the PSII centers were insensitive to the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which may be related to reorganization of the thylakoid membrane. We conclude from these results that PSII does not limit the photosynthetic activity of the cells during the first half of the light period. However, the decline in photosynthetic activity observed during the last half of the light period can be accounted for by limited PSII activity.

Capillary chromatography/microelectrospray mass spectrometry used for the identification of putative cyclin-dependent kinase inhibitory protein in Medicago.

A home-built capillary chromatography/microelectrospray system was used for peptide mass mapping of a putative cyclin-dependent kinase inhibitor protein with molecular mass of 8.5 kDa. The masses of identified tryptic fragments were then input to a protein database search routine. Daughter ion scans were done only on the most abundant tryptic fragments. On the basis of protein database search results and tandem mass spectrometric measurements the bioactive protein was identified as ubiquitin.

Cell cycle phase specificity of putative cyclin-dependent kinase variants in synchronized alfalfa cells.

The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells. Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa). By isolating cDNAs using a cdc2MsA probe, we demonstrate here that at least four additional cdc2 homologous genes are expressed in the tetraploid alfalfa. Proteins encoded by the new cdc2MsC to cdc2MsF cDNAs share the characteristic functional domains of CDKs with the conserved and plant-specific sequence elements. Transcripts from cdc2MsA, cdc2MsB, cdc2MsC, and cdc2MsE genes are synthesized throughout the cell cycle, whereas the amounts of cdc2MsD and cdc2MsF mRNAs peak during G2-to-M phases. The translation of Cdc2MsA/B, Cdc2MsD, and Cdc2MsF proteins follows the pattern of transcript accumulation. The multiplicity of kinase complexes with cell cycle phase-dependent activities was revealed by in vitro phosphorylation experiments. Proteins bound to p13suc1-Sepharose or immunoprecipitated with Cdc2MsA/B antibodies from cells at G1-to-S and G2-to-M phase boundaries showed elevated kinase activities. the Cdc2MsF antibodies separated a G2-to-M phase-related kinase complex. Detection of histone H1 phosphorylation activities in fractions immunoprecipitated with antimitotic cyclin (CyclinMs2) antibodies from G2-to-M phase cells indicates the complex formation between this cyclin and a kinase partner in alfalfa. The observed fluctuation of transcript levels, amounts, and activities of kinases in different cell cycle phases reflects a multilevel regulatory system during cell cycle progression in plants.

Oxidative stress induced by bloodless limb surgery on humans.

Measure of oxidative stress were studied in blood samples from 10 patients undergoing bloodless lower limb surgery. Ischaemia induced a significant increase in plasma hypoxanthine concentration and xanthine oxidase activity both in the operated leg and in the systemic circulation. Five minutes after reperfusion, ratio of xanthine oxidase/total xanthine oxidase and dehydrogenase activities rose moderately, whereas at 20 min xanthine oxidase accounted for all xanthine oxidoreductase activity in the systemic circulation. A significant increase in blood glutathione redox ratio, enhanced oxidation of haemoglobin to methaemoglobin and rise in plasma haemoglobin concentration were present only in the operated limb. Thus, although the level of the potential free radical generators rose significantly both locally and in the systemic circulation, oxidative stress, as indicated by blood glutathione and erythrocyte injuries, remained limited to the reperfused leg.

Follow-up of human papillomavirus (HPV) DNA and local anti-HPV antibodies in cytologically normal pregnant women.

The high level of progesterone during pregnancy may enhance the transcription and replication of genital human papillomaviruses (HPV) through the glucocorticoid/progesterone response element found in the long control region of the viral genome. In this study, cytologically and colposcopically healthy pregnant women were subjected to a follow-up examination. Samples from the uterine cervix were collected during early pregnancy (n = 39), in the third trimester (n = 31), and a few weeks after birth (n = 30). The presence of HPV DNA was detected by polymerase chain reaction (PCR), while local secretory anti-viral IgA antibodies were demonstrated by enzyme-linked immunosorbent assay using synthetic peptide antigens. Follow-up examination by PCR revealed HPV DNA persistence in 5 women. In 5 other cases, HPV positivity changed from negative to positive during the follow-up. There was 1 case which changed from positive to negative and 1 in which the HPV type changed during the study. Altogether, 12 of 39 women (31%) were shown to harbor HPV DNA at some time during follow-up. HPV DNA positivity increased from 18% during early pregnancy to 27% after birth (difference not significant). On the other hand, there was a significant rise in the level of local antibodies against HPV antigens (E2, E7, and L2) between samples collected in early pregnancy and those collected after birth (P < 0.0001). This may indicate the reactivation of genital HPV infections during late pregnancy.

Human papillomavirus DNA and anti-HPV secretory IgA antibodies in cytologically normal cervical specimens.

Cervical specimens collected from 163 cytologically healthy women were screened for the presence of human papillomavirus (HPV) DNA and anti-HPV secretory IgA antibodies. HPV DNA was detected by a general primer mediated polymerase chain reaction (PCR), which amplifies a conserved region from the L1 ORF of genital HPVs. The PCR products were typed by restriction enzyme digestion. A total of 35 samples (21.5%) were positive for HPV DNA (13 samples for HPV 6, 6 for HPV 16, 3 for HPV 18, and 13 for untypeable HPV X). HPV DNA positivity was significantly higher among women under 25 years of age (34.8%) than among the older patients (12.4%) (P < 0.001). An enzyme-linked immunosorbent assay (ELISA) using synthetic peptide antigens was carried out to detect local secretory IgA antibodies against the following HPV specific antigens: HPV 16 E2, HPV 16 E7, HPV 16 L1, HPV 16 L2, and HPV 11 L2. Thirty-four secretions (20.9%) were found to react with at least one of the oligopeptides. Anti-HPV IgA positivity was the highest among women aged 25-32 years, and it was significantly lower in both the younger and the older age groups (P < 0.05). Correlation between HPV DNA and anti-HPV IgA detection was rather weak: anti-peptide IgA positivity was 34.3% (12 of 35) among HPV DNA positive patients compared to 17.2% (22 of 128) among HPV DNA negative women (P < 0.05). The fluctuating course of latent HPV infections should be considered in evaluating the low level of correlation between HPV DNA and anti-HPV IgA positivity.

Oral contraceptive use and human papillomavirus infection in women without abnormal cytological results.

Both experimental and epidemiological data support the idea that oral contraceptive (OC) use may have a stimulating effect to a certain point on cervical carcinogenesis. The current investigation tries to answer the question whether OC use might have an influence on early human papillomavirus (HPV) infections. A total of 425 women without abnormal cytological results were examined colposcopically, and filter in situ hybridisation (FISH) was used to determine the presence of human papillomavirus (HPV) types 6, 11, 16 and 18. Eighty-one cervical specimens (19.1%) were found to be positive for one or more of the HPV types in FISH. HPV positivity was found to correlate with age and parity, being the highest among women under 25 and with less than two births. The use of OCs was inversely correlated with the presence of ectopy or dysplasia in this group of women. On the other hand, HPV positivity was not significantly higher among OC users than among non-users in any colposcopic group. Neither the type of pill used, nor the duration of use had any significant effect on HPV positivity. Further investigations are needed to evaluate the effects of OC use on more severe HPV-induced cervical lesions.

PIP: The authors investigated whether oral contraceptive (OC) use may influence early human papillomavirus (HPV) infections. 425 women aged 18-58 years of mean age 30.1 years with normal cytological results were examined colposcopically, with filter in situ hybridization (FISH) used to determine the presence of HPV types 6, 11, 16, and 18. The women were non-pregnant attendees at a district gynecologic outpatient clinic in Debrecen, Hungary. 81 cervical specimens were found positive for one or more of the HPV types. HPV positivity correlated with age and parity, being the highest among women under age 25 and with less than two births. The use of OCs was inversely correlated with the presence of ectopy or dysplasia in the group, and HPV positivity was not significantly higher among OC users than among non-users in any colposcopic group. Neither the type of pill used, nor the duration of use had any significant effect upon HPV positivity. The authors posit that further investigations are needed to evaluate the effects of OC use upon more severe HPV-induced cervical lesions.

[Experience with total hip prosthesis]. / Totalis csípöendoprotézisekkel szerzett tapasztalataink.

Authors report on their material of total hip endoprosthesis, performed 30 May 1969--31 August 1988 on the Department of Orthopaedics of the Semmelweis University of Medical Science. In this period 1480 operations were made. The formerly and recently used operative technique and the types of the prostheses are described. The intraoperative, the early and the late complications and their treatment are also described. The formerly and recently used tactics of reoperation is reported and attention is called to the fact that the success rate of the replantation can be increased by an adequate technical skill.

[Experience with giant cell tumors]. / Oriássejtes tumorokkal szerzett tapasztalataink.

Authors describe, 64 cases of giant-cell tumour. 5 (9 per cent) was found in the latent, 32 (50 per cent) in the active and 27 (41 per cent) in the aggressive stage when first seen. The operative methods are described and these are determined first of all by the surgical stage of the giant-cell tumour, its size and localization, by the age of the patient and whether primary tumour or a recurrence is found. Based on their experiences, excochleation and filing is suggested in the latent and active stage of this tumour. In tumours in the agressive stage the so called wide resection in the healthy tissue is reasonable. In the rare malignant giant-cell tumour radical resection is suggested. Recurrence was found in 29 per cent after the first operation. Follow-up examinations show that 64 per cent of the patients is symptom free, in 33 per cent the main source of complaints is the consecutive arthrosis, 2 patients were lost in consequence of lung metastasis.

The clinical diagnostic significance of serum antichondrocyte membrane antibodies in osteoarthritis.

In osteoarthritic patients significantly elevated level of antibodies against human chondrocyte membrane extract was found by double solid phase ELISA which seemed to be more specific for the disease than the antibody level of collagen types II, IX and XI. Altogether 86 patients with a mean age of 53.6 years and a ratio of 25% men and 75% women suffering from severe hip joint osteoarthritis were studied in comparison with 44 control persons with a mean age of 35.8 years and identical sex distribution. The specificity of reacting antibodies was proved partly by inhibition test and partly by the fact that the ELISA reaction could be produced only by proteins of the membrane extract bound to collagen (II) after affinity chromatography. The diagnostic importance of the methods is striking, in view of the lack of any other objective laboratory tests for the disease.