Probing the repulsive core of the nucleon-nucleon interaction via the (4)He(e,e'pN) triple-coincidence reaction.

We studied simultaneously the (4)He(e,e'p), (4)He(e,e'pp), and (4)He(e,e'pn) reactions at Q(2)=2(GeV/c)(2) and x(B)>1, for an (e,e'p) missing-momentum range of 400 to 830 MeV/c. The knocked-out proton was detected in coincidence with a proton or neutron recoiling almost back to back to the missing momentum, leaving the residual A=2 system at low excitation energy. These data were used to identify two-nucleon short-range correlated pairs and to deduce their isospin structure as a function of missing momentum, in a region where the nucleon-nucleon (NN) force is expected to change from predominantly tensor to repulsive. The abundance of neutron-proton pairs is reduced as the nucleon momentum increases beyond ∼500 MeV/c. The extracted fraction of proton-proton pairs is small and almost independent of the missing momentum. Our data are compared with calculations of two-nucleon momentum distributions in (4)He and discussed in the context of probing the elusive repulsive component of the NN force.

HLA-G-treated tolerogenic dendritic cells induce tolerogenic potential by increasing expression of B7-1 (CD80) molecules.

BACKGROUND: By consensus, HLA-G has a role in the induction of tolerance via interaction with dendritic cells and manipulation of co-stimulatory molecule expression. The purpose of this study was to demonstrate that HLA-G modified dendritic cells exhibit a decreased ability to stimulate T-cells in vitro due to increased expression of B7-1, which provides regulatory signalling to T-cells as a consequence of binding CD28 and CD152 ligands. METHODS: Bone-marrow cells were cultured from Brown Norway (BN) rat femurs and sorted with anti-rat dendritic cell Ab (OX62) microbeads. Isolated dendritic cells (DCs) were treated with HLA-G tetramer for 3 days. Initially, cells were plated with media, alloantigenic splenocytes, and T-cells and then observed in mixed lymphocyte reaction for thymidine uptake. Also, the cells were analyzed by flow cytometry using antibodies for MHC-II (IA), CD80, and CD86. RESULTS: This study displays that HLA-G-modified DCs decrease induction of an alloproliferative response. In addition, this study demonstrated that HLA-G tetramer treatment decreases CD86 and permits expression of CD80 without altering MHC-II expression. DISCUSSION: This study specifically investigated the role of B7-1 (CD80), showing that HLA-G treatment of immature DCs allows expression of B7-1 (CD80) without altering MHC-II expression and simultaneously decreases B7-2 (CD86) expression. Therefore, a low level of B7-2 (CD86) allows attenuation of T-cell response after activation, both of which are beneficial to immune tolerance. The selective expression of B7-1 (CD80) is important and may serve as a guide for future therapies aimed at transplant tolerance.

[Effect of rocuronium on the diaphragm, musculus adductor pollicis and orbicularis oculi in two groups of different age]. / Neuromuskuläre Wirkzeiten von Rocuronium am Diaphragma, Musculus adductor pollicis und orbicularis oculi in zwei Altersgruppen.

OBJECTIVE: Onset time and recovery from non depolarising neuromuscular blockade depends on the tested muscle and is influenced by the age of the patient. This study compares the neuromuscular blocking effect of rocuronium on the diaphragm, adductor pollicis and orbicularis oculi muscle in young and elderly patients. METHODS: After institutional ethics committee approval and written informed consent, 20 adult patients (ASA I - II), age 18 - 59 and > 65, have been included. Neuromuscular response was measured by accelerography for the adductor pollicis and orbicularis oculi muscle. Monitoring of the diaphragm consisted of measurement of the airway pressure against an occluded tracheal tube during magnetic phrenic nerve stimulation. Onset time and recovery were measured after injection of 0.6 mg/kg Rocuronium. RESULTS: The adductor pollicis had the fastest onset time ( young 2.3 min, old 2.2 min), followed by diaphragm ( young 3.6 min, old 3.4 min) and orbicularis oculi muscle ( young 3.7 min, old 4.8 min). There was a complete blockade of the diaphragm in 50 % of all patients (Adductor pollicis 100 %, orbicularis oculi 40 %). Neuromuscular recovery, recovery index and TOF 0.8 differed significantly between young and elderly patients. Onset of recovery was earlier at the diaphragm ( young 15.9 min, old 22.0 min) compared to the peripheral muscles (adductor pollicis young 25.6 min, old 37.9 min, orbicularis oculi young 23.8 min, old 27.5 min). CONCLUSION: 2 fould ED95 of rocuronium often results in an incomplete neuromuscular blockade of the diaphragm. Therefore monitoring of the peripheral muscles in patients given a single dose of rocuronium often overestimates the degree of diaphragmatic relaxation, but is a save predictor of recovery. Especially in elderly patients were prolonged neuromuscular blockade should be expected, a neuromuscular monitoring is recommended.

Decreased proliferation of human melanoma cell lines caused by antisense RNA against translation factor eIF-4A1.

Control of translation initiation was recognised as a critical checkpoint for cell proliferation and tumorigenesis. In human melanoma cells, we have previously reported consistent overexpression of translation initiation factor eIF-4A1. Here, we investigated by transfection of antisense constructs its significance for the control of melanoma cell growth. The tetracycline-inducible expression system was established in melanoma cells, and three fragments of the 5'-, central-, and 3'-portion of the eIF-4A1 cDNA were subcloned in antisense and in sense orientation after a tetracycline inducible promoter. Significant proliferation decrease was obtained after transient transfection and induction of antisense RNA directed against the 5'- and the central portion (up to 10%), whereas, no effects were seen after induction of the 3'-fragment and the sense controls. Cell clones stably transfected with the central antisense fragment revealed after doxycycline induction reduced expression of endogeneous eIF-4A1 mRNA correlated with decreased proliferation rates (up to 6%). These data demonstrate the applicability of antisense strategies against translation factors in melanoma cells. Translation initiation factor eIF-4A1 contributes to the control of melanoma cell proliferation and may be taken into consideration when scheduling new therapeutic approaches targeting the translational control.

Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells.

Several techniques for the enrichment of nucleated fetal red blood cells present in maternal blood have been reported. Here we describe the use of a quantitative fluorescence in situ hybridization (FISH) method and in vitro suspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatography (HPLC) method, that allows us to determine as few as 100 cells containing haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 cells each) on microscopic slides, and inclusion of internal controls to monitor the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of peripheral blood from 26 pregnant women were grown in media containing growth factors and hormones to yield over 10(7) of immature erythroid cells within two weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A total of approximately 8x10(8) maternal cells were added into tissue culture medium for these 13 cultures, resulting in about 2x10(8) nearly pure erythroid cells after two weeks. Whereas fetal cells, alone or added into cultures of peripheral blood, grow rapidly and can be detected quantitatively, we could not find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) peripheral blood cells probed by FISH analysis (half of which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be established routinely and easily. With the quantitative FISH technique used, 750 000 cells per slide can be screened reliably for cells with Y chromosomes. However, the stringent quality-criteria and most elaborate methods indicate that fetal cells in maternal peripheral blood can not be found using the current technology.

Relevance of zinc in human sperm flagella and its relation to motility.

OBJECTIVE: To measure the zinc content of human sperm flagella and to analyze its relation to sperm motility. DESIGN: Prospective study. SETTING: Center of Dermatology and Andrology. PATIENT(S): Semen samples collected from 90 andrology patients and healthy donors after 3-5 days of sexual abstinence. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm morphology after Shorr staining, sperm motility, and patient age were recorded. In addition, zinc concentrations in the seminal plasma, sperm heads, and flagella were determined with the use of atomic absorption spectrometry. RESULT(S): The mean zinc concentration was 144.3 mg/L in the seminal plasma and 146.9 mg/L in the whole ejaculate and was significantly correlated with parameters of motility. The sperm heads contained only 6.7% of the zinc that was present in the whole spermatozoon. The zinc concentration in the flagella was negatively correlated with sperm motility and velocity. In addition, it was positively correlated with the percentage of abnormally blue-stained flagella and the age of the patients. CONCLUSION(S): Our results clearly demonstrate the importance of zinc elimination during epididymal sperm maturation for functional competence of the outer dense fibers and, therefore, generation of motility.

Restriction digest PCR (RD-PCR) for the analysis of gene mutations. Application to Ki-ras.

The Kirsten-ras (onco)gene codes for a GTP-binding membrane protein that is involved in signal transduction. Activated ras triggers a cascade of protein-phosphorylations that ultimately lead to cell proliferation. Ras-mutations are the main cause for adenocarcinomas of the pancreas besides some mutations in the tumor suppressor gene p53 and the c-erbB-2 oncogene. The site of ras mutations in pancreatic cancer is restricted to codon 12 that normally encodes a glycine. For analysis of codon-12 mutations, DNA is extracted from cells in pancreatic fluid and amplified by PCR. Because most of these cells originate from normal tissue with only a few tumor cells in the fluid, "enrichment PCR" must be utilized: In a first round of the PCR, ras sequences from all cells are amplified. By utilizing an appropriate restriction enzyme, wild-type sequences can be digested and the remaining fragments containing mutated sequences be amplified again. An artificial restriction site must be introduced by the 5'primer (...GGA CCT GGT...) for an enzyme (BstNI) (5'CC!WGG 3') to differentiate between wild-type sequence (...GGA GCT GGT...) (during amplification, the G is replaced by a C) and mutated sequences (_...GGA GCT (GTT), (CGT), (CCT), etc.). The necessary manipulations pose a considerable risk for contamination for the second round of the PCR procedure. Therefore, we considered whether it would be feasible to perform the restriction digest simultaneously with the first PCR reaction, and avoiding the second round altogether. The results of our experiments demonstrate that one tumor cell in 1000 normal cells can be determined readily, paralleling the results with the original two step-assay. The restriction enzyme used to enrich mutated sequences is stable long enough to be included into the PCR procedure. By this, wild-type sequence amplicons are digested while they are formed and mutated sequences can be enriched selectively.

In vivo deuteration of transfer RNAs: overexpression and large-scale purification of deuterated specific tRNAs.

Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.

The elongating ribosome: structural and functional aspects.

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.

Contributions to the taxonomy and biology of Clostridium difficile.

Clostridium difficile was incriminated by Hughes and Jarvis (1987) as a cause of intestinal infections in USA in the 1980-1984 period in 45 p. 100 of cases, whereas Salmonellae only in 12 p. 100. Four strains of this organism are studied in this paper in comparison with ten strains of C. bifermentans and six of C. sordellii, because the three species share a common antigen and have other common characteristics, as well. However, spores of C. difficile swell the bacteria and those of other bacteria (C. bifermentans and C. sordellii) do not; C. difficile does not produce indole, whereas the other species produce it. We confirmed the selective capacity of the medium of George et al. (1979) using the "alcohol shock" and as selective agents cycloserine D and cefoxitin. C. difficile proved to be most susceptible to metronidazole and rifampin. Whereas the former antibiotic was considered as a cause of post-antibiotic intestinal infections by different authors, the second was not, to our knowledge. The strain 10463 has a considerable toxicity (1000 DLM/ml for the white mouse, and pathogenicity--2000--5000 DCL for the white mouse, as compared to 25 DCL of the other three strains). Using this toxin an antitoxic serum was obtained in horse, with a capacity of neutralizing the action of the toxin up to a dilution of 1 p. 1000.

[The timely diagnosis of postoperative clostridial infection]. / Diagnosticul în timp util al infectiei clostridiene postoperatorii.

Authors report a case of clostridial myonecrosis postcholecystectomy. The causes of the occurrence of this severe, frequently lethal complication are reviewed, as well as the difficulties of an early diagnosis, the only factor able to save the patient. The curative treatment of the detected infection requires the initiation of a series of local and general measures and a permanent cooperation between the surgeon, the specialist in resuscitation and the infectionist.

Detection of Bacteroides fragilis group by immunofluorescence.

The following strains: B. fragilis subspecies thetaiotaomicron (A); B. fragilis subspecies fragilis strain E-1, E-2, M, St., Se., Ni., 8, 16; B. fragilis subspecies distasonis 145 (D) were serologically studied by immunofluorescence as compared to agglutination. Anti-B. fragilis sera titration by immunofluorescence (IF) reaction, as compared to agglutination reaction in tube, was more sensitive (2-16 times higher titers), specific and reproducible. Among the organisms from B. fragilis group, species, subspecies and even train specificity was noticed. Also, the possibility for rapid identification of anaerobic organisms, belonging to B. fragilis group, in pathologic products obtained from experimentally infected animals (mice and rats), by IF reaction, in comparison with classic methods (anaerobic cultures and biochemical determinations) was studied. Of 87 studied animals, 61 proved positive by cultures and 59 by IF; 56 animals were shown positive and 23 animals proved negative by both methods (intermethods concordance in 79 cases). Statistical analysis of IF results provided the following: method sensitivity (detection capacity of real-positive cases)-91.80%; method specificity (detection capacity of real-negative cases)-88.46%; false-positive cases-11.53%; false-negative cases-8.19%. Immunofluorescence proved specific, sensitive, practical and rapid method detection of non-sporulated anaerobic organisms species and subspecies belonging to Bacteroides fragilis group.

[A case of Caffey-Silverman syndrome in a 3.5 month old infant]. / Przypadek zespolu Caffeya-Silvermana u 3,5-miesiecznego niemowlecia.

The authors presented a case of Caffey-Silverman syndrome in 3.5 months old infant with the lesions only in mandible.