Profound isotope effect in dissociation of triatomic hydrogen.

Three-particle dissociation of high-lying Rydberg states of D3 is induced by an external electric field. We observe that the momentum vector correlation map of the center-of-mass motion of the fragments converges near the ionization threshold to two distinct fragment configurations, the near linear geometry and the symmetric acute angle geometry. A comparison is made with the momentum vector correlation map recorded in dissociative recombination of D3(+) with slow electrons and with the corresponding results for H3 where the acute angle geometry is conspicuously absent.

The use of xenografts to prevent inferior border defects following bilateral sagittal split osteotomies: three-dimensional skeletal analysis using cone beam computed tomography.

The aim of this retrospective study was to investigate grafting in the osteotomy gap during bilateral sagittal split osteotomy (BSSO), using a xenograft and fibrin glue. Hard tissue defects in the inferior mandibular border were assessed using cone beam computed tomography scans taken 1 week and 1year postoperatively. The study group of 20 patients underwent bone grafting during BSSO (mean age 26.1years; mean horizontal displacement 8.5mm) and the control group of 20 patients did not (mean age 30.2 years; mean horizontal displacement 7.6mm). The mean height of the mandibular defects was significantly lower in the study group, but there was no significant difference in volume measurements between the groups. Grafting had a negligible effect on large displacements (9.0-15.0mm), which might have been due to an inadequate amount and/or positioning of the graft, or to poor dimensional stability. This may be resolved by improved graft positioning or by using a different kind of (xeno)graft.

Group IIA and group V secretory phospholipase A(2): quantitative analysis of expression and secretion and determination of the localization and routing in rat mesangial cells.

Mesangial cells can be induced to express group IIA and group V secretory phospholipase A(2) (sPLA(2)) at the mRNA level and at the protein level. In this report we quantitatively analyze the expression of both proteins in stimulated cells by Western blot techniques. We found that 75-80% of the total amount of synthesized group IIA sPLA(2) was secreted. The synthesized group V sPLA(2), however, was present almost exclusively intracellularly. The amount of group V present in the cell was comparable to the intracellular amount of group IIA sPLA(2). We furthermore studied the localization and routing of both proteins. Using fusion proteins of the group IIA or group V pre-sPLA(2) with green fluorescent protein it was established that both presequences are able to direct the proteins to the Golgi system. In immunofluorescence studies group V sPLA(2) expressed by rat mesangial cells was located in a punctate pattern in the cytosol with an enrichment near the nucleus. Immunofluorescent confocal laser scanning microscopy revealed that the group V and IIA sPLA(2) show partial colocalization in a Golgi-like structure in the inner part in the cell, but no colocalization was seen in the vesicles in the cytoplasm. The images also showed that group IIA sPLA(2) was located throughout the cell while group V was mainly present in the inner part of the cell. After treatment of the cells with brefeldin A or monensin the group IIA enzyme could no longer be detected, while group V sPLA(2) was still present although its localization was somewhat dependent on the treatment. Collectively, these results indicate that the two enzymes differ in both localization and routing in the cell, which underscores the hypothesis that the enzymes might have different functions.

Regulation of the expression of group IIA and group V secretory phospholipases A(2) in rat mesangial cells.

Rat mesangial cells synthesize and secrete a secretory phospholipase A(2) upon stimulation of the cells with cytokines, like IL-1beta and TNF and with cAMP elevating agents like forskolin. This enzyme was previously characterized to belong to group IIA sPLA(2). The discovery of several other low molecular weight phospholipases, like group IIC in murine testis and group V in human and rat heart, prompted investigations on the presence of group IIC and group V sPLA(2) in rat mesangial cells. This was done by isolating the RNA from stimulated cells and performing RT-PCR, using primers specific for group IIC and V sPLA(2). The results indicate that rat mesangial cells upon stimulation express next to group IIA also group V sPLA(2). No indications were obtained for the expression of group IIC sPLA(2). The regulation of the expression of group V sPLA(2) at the mRNA level was further investigated by examining the time-dependent expression, the influence of dexamethasone and the signaling route of the IL-1beta stimulation. The results show that the IL-1beta induced expression of group V sPLA(2) mRNA was time dependent and, similar to that of group IIA sPLA(2) mRNA, involves activation of NF-kappaB. However, in contrast to the group IIA sPLA(2), the expression of group V sPLA(2) was not influenced by the presence of dexamethasone. The expression of both phospholipases was also examined at the protein level in stimulated mesangial cells. Western blot analysis shows that stimulated mesangial cells synthesize both group IIA and group V sPLA(2) protein but the expression of group V is lower compared to that of group IIA sPLA(2). In addition, the extent of secretion into the medium appears to be considerably higher for group IIA than for group V sPLA(2).

Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A(2).

During recent years, the high phospholipase A(2) (PLA(2)) concentrations at sites of inflammation and in circulation in several life-threatening diseases, such as sepsis, multi-organ dysfunction and acute respiratory distress syndrome, has generally been ascribed to the non-pancreatic group IIA PLA(2). Recently the family of secreted low molecular mass PLA(2) enzymes has rapidly expanded. In some cases, a newly described enzyme appeared to be cross-reactive with antibodies against the group IIA enzyme. For this reason, reports describing the expression of group IIA PLA(2) during inflammatory conditions need to be reevaluated. Here we describe the identification of the PLA(2) activity in sera of acute chest syndrome patients and in sera of trauma victims. In both cases, the PLA(2) activity was identified as group IIA. This classification was based upon cross-reactivity with monoclonal antibodies against group IIA PLA(2) which do not recognize the recombinant human group V enzyme. Moreover, purification of the enzymatic activity from the two sera followed by N-terminal amino acid sequence analyses revealed only the presence of group IIA enzyme.

Enzymatic properties of rat group IIA and V phospholipases A(2) compared.

Group IIA and V phospholipases A(2) (PLA(2)s) are known to play a role in inflammatory responses. We have constructed a bacterial expression vector for rat group IIA and V PLA(2)s, over-expressed, folded and purified the proteins with the aim to study and compare the properties of the enzymes in detail. For zwitterionic phospholipid micelles, both enzymes display optimum activity at pH 8. 0 and absolutely require Ca(2+) for enzymatic activity. In the presence of substrate, group V PLA(2) has a high affinity for Ca(2+) (K(Ca2+)=90 microM) while K(Ca2+) of group IIA PLA(2) was found to be 1.6 mM. The absence of substrate only marginally influences the Ca(2+) affinities. In contrast to group IIA PLA(2), group V PLA(2) does not show a jump in the activity profile at substrate concentrations around the critical micelle concentration. Direct binding studies using n-alkylphosphocholines indicate that group V PLA(2) forms protein-lipid aggregates at pre-micellar lipid concentrations in a cooperative and Ca(2+)-dependent manner. This behavior, which is comparable to that observed for the PLA(2) from Naja melanoleuca snake venom, reflects the high affinity of this enzyme for zwitterionic phospholipids. Competitive inhibition by the substrate analogues (R)-2-dodecanoylaminohexanol-1-phosphocholine and its phosphoglycol derivative was tested on zwitterionic micelles as substrate. Group IIA PLA(2) shows a preference for the phosphoglycol inhibitor whereas the phosphocholine inhibitor binds stronger to the active site of group V PLA(2). The enzymatic activity was also measured on zwitterionic liposomes which appear to be much better substrates for group V PLA(2) than for group IIA PLA(2). The overall results suggest that group V PLA(2) is better suited for action on biological membranes than group IIA PLA(2).

Spinocerebellar degeneration: hexosaminidase A and B deficiency in two adult sisters.

Two adult sisters had spinocerebellar degeneration. Biochemical studies revealed a very low activity of both fraction A and fraction B of the lysosomal enzyme, hexosaminidase, in serum and leukocytes. A skin biopsy showed lesions suggestive of neuronal storage disease. The disorder seems to be an adult form of GM2 gangliosidosis.

Suppression of the development of experimental allergic encephalomyelitis by suramin.

Suramin was tested for its ability to suppress experimental allergic encephalomyelitis. Prophylactic administration caused significant reduction in the severity of the disease, incidence of paralysis and cellular infiltration in nervous tissue. Therapeutic treatment with suramin also caused a reduction in the severity of the disease, the incidence of paralysis and cellular infiltration, but to a lesser extent. Significantly fewer animals were paralysed for more than two days on therapeutic treatment.

In vitro inhibition of the classical pathway of human complement by a natural microbial product, colistin sulphate.

Colistin sulphate was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC14 formation from EA and C2-deficient serum, EAC1-7 formation from EAC1-3, C5, C6 and C7 and the interaction of EAC1-7 with C8 and C9, though less efficiently. It did not inhibit formation of C3/C5 convertase of the alternative pathway. The inhibition of the classical pathway was reversible since hemolytic activity was completely restored after dialysis.

In vitro inhibition of the classical pathway of human complement by polymyxin B.

Polymyxin B was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC1-9 formation from EAC1-3 and C5-9 though slightly less efficiently. It did not inhibit C3/C5 convertase of the alternative pathway or its formation. The inhibition of the classical pathway was reversible since hemolytic activity was almost completely restored after dialysis.

Does private religious activity prolong survival? A six-year follow-up study of 3,851 older adults.

BACKGROUND: Previous studies have linked higher religious attendance and longer survival. In this study, we examine the relationship between survival and private religious activity. METHODS: A probability sample of elderly community-dwelling adults in North Carolina was assembled in 1986 and followed for 6 years. Level of participation in private religious activities such as prayer, meditation, or Bible study was assessed by self-report at baseline, along with a wide variety of sociodemographic and health variables. The main outcome was time (days) to death or censoring. RESULTS: During a median 6.3-year follow-up period, 1,137 subjects (29.5%) died. Those reporting rarely to never participating in private religious activity had an increased relative hazard of dying over more frequent participants, but this hazard did not remain significant for the sample as a whole after adjustment for demographic and health variables. When the sample was divided into activity of daily living (ADL) impaired and unimpaired, the effect did not remain significant for the ADL impaired group after controlling for demographic variables (hazard ratio [RH] 1.11, 95% confidence interval [CI] 0.91-1.35). However, the increased hazard remained significant for the ADL unimpaired group even after controlling for demographic and health variables (RH 1.63, 95% CI 1.20-2.21), and this effect persisted despite controlling for numerous explanatory variables including health practices, social support, and other religious practices (RH 1.47, 95% CI 1.07-2.03). CONCLUSIONS: Older adults who participate in private religious activity before the onset of ADL impairment appear to have a survival advantage over those who do not.

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.

Comparison of the spectrophotometric determination and the two-stage coagulation assay of tissue factor activity.

The tissue factor activity of human brain thromboplastin and 6 commercial thromboplastins was determined by a spectrophotometric method and a two-stage coagulation assay. The thromboplastins were incubated with an excess of Factor VII and Factor X, and the activation of Factor X was estimated from the rate of hydrolysis of the chromogenic substrate S-2222 and from the coagulation time of plasma enriched in phospholipids. The results obtained by the two methods were related linearly and showed a correlation coefficient of 0.89. The coefficient of variation was 11% in the spectrophotometric method and 25% in the two-stage coagulation assay.

The significance of the estimation of serum myoglobin in neuromuscular diseases.

The serum myoglobin concentration was determined and compared with the serum creatine kinase activity in 230 patients suffering from various neuromuscular diseases. No correlation was found between the two levels. In general serum creatine kinase activity estimation seemed more sensitive for the detection of neuromuscular disease than serum myoglobin estimation. In myotonic dystrophy, however, determination of serum myoglobin was distinctly the more sensitive method.

An improved method for the concentration of cerebrospinal fluid cells by suction tip and sedimentation chamber.

To concentrate cells from cerebrospinal fluid, a new method was developed. The technique is a modification of the cell sedimentation method of Sayk. The fluid is absorbed after passing through a Nuclepore filter, pore size 0.4 micron. With the aid of simple utensils it is possible to obtain a cell preparation of high quality using only 0.3 ml of cerebrospinal fluid. The cytomorphology is as good as that achieved by Sayk's sedimentation technique. The cell yield was determined using an accurate and precise method to estimate the number of cells present in the cerebrospinal fluid. The average cell yield is 90%, and all staining methods are applicable. The system is easily modified, and can be suited to many other fluids and purposes.

The beta 2-microglobulin content of the cerebrospinal fluid in neurological disease.

Beta 2-Microglobulin levels in the CSF and serum of 125 neurological patients were determined. The concentration of beta 2-microglobulin in CSF and serum is approximately the same, ranging from 0.6--2.3 mg/l. No clear relationship could be found between the CSF beta 2-microglobulin level on the one hand and the total protein content and white cell count on the other. There was no clear relationship between the serum beta 2-microglobulin and CSF microglobulin content. An increase of beta 2-microglobulin in CSF was found most often in cases of serious infection of the meninges or of the central nervous system. An elevated beta 2-microglobulin content can occur as the only abnormal feature of the CSF.

Central nervous system involvement in early and late syphilis: the problem of asymptomatic neurosyphilis.

Patients with syphilitic infections are at risk of development of symptomatic neurosyphilis. Adequate treatment with 2.4-7.2 x 10(6) units benzyl penicillin-G intramuscularly within 1 year after infection will rule out this risk. However, more than 1 year after infection this treatment is not fully reliable. In asymptomatic CNS involvement (asymptomatic neurosyphilis) only intravenous penicillin treatment is considered to be adequate in the prevention of neurosyphilis. In this study we redefined criteria for this condition by comparing serum and cerebrospinal fluid (CSF) samples of symptomatic neurosyphilitic patients with those of latent syphilitic patients without CNS involvement. Diagnostic criteria of the World Health Organization and of Centers of Disease Control for asymptomatic neurosyphilis (positive CSF Venereal Disease Research Laboratory (VDRL) test, combined with raised CSF cell count and/or protein content) were studied and compared with some newer parameters such as signs of intrathecal treponemal antibody production (Treponema pallidum haemagglutination assay and intrathecal Treponema pallidum assay index), immunoglobulin G (IgG) and M (IgM) index. The results of this study in 203 syphilitic patients revealed that either a positive CSF-VDRL or combination of a raised IgG and/or IgM index with an elevated CSF cell count both are useful criteria for "ruling-in" asymptomatic neurosyphilis.

Differential diagnosis of genetic disease by DNA restriction fragment length polymorphisms.

DNA restriction fragment length polymorphisms (RFLPs) are used for diagnosis of genetic disease in families known to be affected by specific disorders, but RFLPs can be also useful for the differential diagnosis of hereditary disease. An RFLP pattern represents the inheritance of chromosomal markers in a family, and such a pattern may be compatible with the inheritance of a certain disorder in that family. Probabilities to find such a pattern if the disorder were present and if it were absent can be combined with the prior probabilities of disease considered in the differential diagnosis on the basis of previous clinical and laboratory data. Bayes' theorem is used to calculate the posterior probabilities of the diseases in question. This approach is illustrated in a family suffering from either spinal muscular atrophy (an autosomal recessive disease) or Becker muscular dystrophy (an X-chromosomal disorder). Probabilities to exclude a certain disorder can be calculated in advance, as some RFLP patterns are not compatible with the presence of that disorder.

Reinvestigations into the formation and assay of C3bBbP complexes.

C3bBbP complex formation was studied by an enzyme linked immunosorbent assay (ELISA). Microtitre plates were coated with anti-P to trap the complexes and peroxidase labelled anti-C3 was used to detect them with the help of substrates of peroxidase. Incubation of normal serum pool (NSP) at 37 degrees C in the presence of high concentrations (greater than or equal to 0.5 mmol/l) of Mg2+, usually used in alternative pathway (AP) assay systems, caused the generation of C3bBbP complexes. This generation was not observed when NSP was incubated in the presence of low Mg2+ concentration (less than or equal to 0.2 mmol/l) or EDTA. The concentration of Mg2+ required for maximum complex formation was 2.0 mmol/l under the experimental conditions. Complexes could not be generated in B-depleted serum. Incubation of NSP with endotoxin or CoVF in the presence of 0.2 mmol/l Mg2+ caused the generation of the complexes. The generation was influenced by ionic strength in the incubation mixture. Endotoxin and Mg2+-dependent generation of complexes could not be detected when peroxidase-labelled anti-B was used instead of peroxidase-labelled anti-C3. Serum incubated with 0.2 mmol/l Mg2+ or EDTA apparently detected in vivo formed complexes whereas that incubated with 0.2 mmol/l Mg2+ and endotoxin reflected the complex forming capacity of the serum. The serum of a patient with Raynaud's phenomenon having 45% of normal AP activity did not show increased amounts of preformed complexes but had the ability to generate the complexes to a level of about 45% of that attainable by NSP. These observations suggest that the ELISA used here has the potential of detecting activation as well as the integrity of the AP under carefully controlled conditions.

Immunoglobulin D in cerebrospinal fluid.

Immunoglobulin D (IgD) was measured by radioimmunoassay in paired cerebrospinal fluid and serum samples from patients with various neurological diseases. The IgD index was calculated for every patient and compared with the IgG index. An increased IgD index was found in 18 out of 41 patients suffering from multiple sclerosis and in 52 out of all 122 patients investigated. An increased IgD index did not always coincide with an increased IgG index. An increased IgD index suggests an abnormal intrathecal synthesis of this immunoglobulin within the central nervous system. We conclude that the determination of IgD in the cerebrospinal fluid yields additional information on immunological reactions within the central nervous system.