Search for substrates for prolyl oligopeptidase in porcine brain

The function of prolyl oligopeptidase (PO) has been associated with several disorders of the central nervous system. The purpose of this study was to identify endogenous substrates for recombinant porcine PO in porcine brain. The smaller polypeptides were extracted from total brain homogenates and fractionated by two-dimensional chromatography prior to incubation with PO. Shifts in the mass spectrum between the control and the incubated sample, marked potential substrates. Using MSMS peptide sequencing techniques, we identified several fragments of intracellular proteins as potential substrates, which opens new perspectives for finding the function of PO in the intracellular space.     

Guidelines for indications for coronary revascularization in The Netherlands

In 1996 the Minister of Public Health, Welfare and Sports in The Netherlands published a 'Planning Decree Special Interventions in the Heart'. She requested from the professional organizations guidelines for the indications for interventions in the heart. A working group was formed with representatives from the Dutch professional organizations for cardiology and thoracic surgery, to address this issue for patients with coronary artery disease. The working group confirmed the need to discuss all patients who are considered for either elective or emergency revascularization during a multidisciplinary consultation in (or with) one of the specialized Dutch hospitals. During this meeting of the 'heart team', at least one interventional cardiologist and one thoracic surgeon should be present. There are three possible outcomes of the heart team's consultations for each patient: drug therapy only ('conservative management'), coronary surgery or catheter intervention. For each case, the team should indicate the expected benefit, the risk of the intervention, the urgency and the estimated waiting time. The guidelines presented in this paper address these issues for three patient categories: stable angina pectoris, unstable angina pectoris and acute myocardial infarction.     

Evidence for a protective role for adiponectin in osteoarthritis

Obesity has been associated with an increased risk of osteoarthritis (OA). However, the mechanism by which obesity contributes to OA remains uncertain. Adiponectin, an adipocyte-derived hormone, has shown anti-diabetic and anti-atherogenic properties. In the present study, we aimed to investigate the potential role of adiponectin in OA disease. We demonstrated that adiponectin was present in OA synovial fluid (SF) and its expression level was almost 100-fold decrease compared with that in OA plasma. FPLC and ELISA studies revealed the distribution and abundance of the adiponectin complexes in plasma and SF from patients with OA. The percentage of high molecular weight (HMW) per total adiponectin in OA SF was lower than in OA plasma, while that of the hexamer form was similar and the trimer form was higher. The expression levels of adiponectin receptors AdipoR1 and AdipoR2 were examined in human OA tissues by RT-PCR. AdipoR1 was abundantly expressed in cartilage, bone and synovial tissues, whereas AdipoR2 was rarely detected. Finally, the effects of adiponectin on primary chondrocyte functions were studied by using antibody-based protein array and RT-PCR. The patterns of mRNA expression and protein production strongly indicate that adiponectin is involved in the modulation of cartilage destruction in chondrocytes by up-regulating TIMP-2 and down-regulating IL-1beta-induced MMP-13. Together these findings clearly indicate that the adiponectin may act as a protective role in the progression of OA, and this also provide new thinking on the relationship between obesity and OA.     

A mass-balance model for phosphorus in lakes accounting for biouptake and retention in biota

SUMMARY 1. A new mass-balance model was developed to predict phosphorus fluxes to, within and from lakes and lake concentrations of phosphorus.
2. The model is driven by data easily accessed from standard monitoring programmes or maps, so it should be useful in many contexts of lake management.
3. The phosphorus model gives seasonal (weekly) variations and accounts for all fundamental abiotic/biotic interactions and feedbacks for lakes in general for phosphorus. The model has been developed within the framework of a more comprehensive ecosystem model, LakeWeb, which accounts for production values and biomasses of nine functional groups of organisms (phytoplankton, bacterioplankton, herbivorous and predatory zooplankton, prey and predatory fish, zoobenthos, macrophytes and benthic algae).
4. The LakeWeb-model has been critically tested in a wide limnological domain and shown to predict very well. By using this model, it is possible for the first time to predict realistic values of biouptake and retention of phosphorous in biota in lakes on a weekly basis.     

Higher permeability for water than for ethyl alcohol in Nitella

If we apply water at one end of a Nitella cell, A, and place at the other end, B, a solution of a substance which does not penetrate, such as sucrose, water enters the cell at A, passes along inside the cell, and escapes at B. But if in place of sucrose we use a substance which penetrates such as ethyl alcohol the flow of water is lessened and this fact makes it possible to measure the amount of alcohol which enters. (An increase in the size of cells placed in solutions of alcohol does not necessarily indicate that the number of mols of alcohol entering is greater than the number of mols of water leaving the cell.) The permeability for water is more than 18 times as great as for ethyl alcohol. The behavior of the 2 substances was compared in the same individual cell with a driving force which at the start was the same for both substances. The number of mols entering per second per cm.² of surface with a driving force of 1 atmosphere at 25°C. is 0.772 (10^–6) for water and 0.042 (10^–6) for ethyl alcohol. The experiments indicate that the non-aqueous substance at the surface of the protoplasm has a higher partition coefficient for water than for ethyl alcohol, although the protoplasmic surface is composed of materials not miscible with water.     

蝴蝶兰EMS离体化学诱变及再生植株RAPD检测

用EMS对离体培养的蝴蝶兰类原球茎薄切片进行化学诱变,研究了不同浓度、不同时间诱变处理对类原球茎薄切片生长、类原球茎再生及再生苗生长的影响,并对再生苗DNA进行了RAPD检测。结果表明,诱变剂EMS对类原球茎薄切片生长产生重要影响,部分薄切片褐化死亡,再生类原球茎生长受到抑制,再生苗数量减少,表现出明显的伤害作用,这种伤害作用随诱变剂浓度的加大和处理时间的延长而加重。诱变剂EMS使部分再生苗畸变,叶片皱缩、变厚。并产生2株叶形、叶色突变体。部分再生苗表现出RAPD图谱带型的多态性,暗示DNA序列发生了一定的变异。EMS适合诱变剂量为浓度0．4％处理2d～4d。     

Evidence for Cometabolism in Sewage

A procedure was developed to demonstrate cometabolism in models of natural ecosystems. The procedure involves showing the formation of metabolic products in high yield and the lack of incorporation of substrate carbon into cellular constituents. Samples of four ¹⁴C-labeled herbicides (trifluralin, profluralin, fluchloralin, and nitrofen) were incubated with sewage aerobically and under discontinuous anaerobiosis for 88 days, and fresh sewage was added at intervals. Products were formed from each of the herbicides in nonsterile, but not in sterile, sewage. The yield of recovered products reached 87% for profluralin and more than 90% for fluchloralin and trifluralin, and the number of products ranged from 6 for nitrofen to 12 for fluchloralin. Concentrating the sewage microflora 40-fold greatly enhanced the rate of conversion. None of the radioactivity from the herbicide entered the nucleoside pool of the sewage microflora. The lack of incorporation of substrate carbon into cells and the almost stoichiometric conversion of the substrate to organic products indicate that members of the microbial community were cometabolizing the test compounds.     

A capacity for mixing in capillary wells for microplates

Sample/reagent mixing is important in microplate instrumentation and the approach of mixing prior to dispensation into the wells can be problematic when assays involve a mixture of different proportions of reagents. We demonstrate here with capillaries, a simple and highly effective method that uses air actuation and liquid surface tension to create pendant drops in which the inherent inner circulatory flow within accomplishes mixing. The approach portends the capability to be incorporated into microplates in order to provide a useful feature for assay investigations.     

Assessment of Needs for Plasma for Fractionation in Europe

In the early 1990s, a series of outbreaks of hepatitis C (HCV) infections clustering among recipients of certain lots of plasma-derived medicinal products (PDMP) alarmed regulatory authorities, manufacturers and the public alike. Also, a few episodes of Hepatitis A (HAV) infections occurred in haemophiliacs receiving solvent-detergent-treated factor VIII concentrates. Thus, several measures were brought into effect to reestablish the safety of the incriminated products and to further increase the margin of safety of PDMP in general. Therefore, intramuscular immunoglobulins had to be free of HCV RNA as shown by nucleic acid amplification technology (NAT) in the final products. Furthermore, the manufacturing process of PDMP had to be validated for both viral inactivation and elimination. Finally, HCV-NAT was to be standardised and implemented as a validated test of plasma pool samples.In 1994, a joint meeting of EPFA, EAPPI and Regulatory Authorities was held in Brussels to outline the state of the art and to delineate the actions to be taken. Five years later, in 1999, the incidence rates of HIV, HBV and HCV in unpaid blood donors have been minimized, especially in European countries. With probabilities for window period donations as low as 0.6 in 1 million for both HIV and HCV and 2.1 in 1 million for HBV in Switzerland, labile blood products have reached extreme, but not absolute safety. The introduction of HCV-NAT roughly doubles this safety resulting in a 1 in 3 million probability of a window donation.Concomittantly, extensive viral validation studies document effective inactivation and removal of viruses in PDMP. The demonstrated margins of safety, expressed as logarithmical reduction factors (LRF), range from 4 to over 20 log(10), depending on product, virus, and inactivation procedure used. Further progress to even safer PDMP shall be acomplished by consolidating the GMP processes, abandoning of obsolete requirements and harmonising national regulations within Europe. Before introducing new measures for additional agents such as HAV or Parvovirus B 19, gains and risks and even potential new threats have to be carefully assessed. Alternative efforts for the safeguard of patients, e.g. vaccination for HAV, need to be balanced against the risks of changing established and validated manufacturing procedures of PDMP with long-lasting safety records.     

Cross-validation analysis for genetic evaluation models for ranking in endurance horses

Ranking trait was used as a selection criterion for competition horses to estimate racing performance. In the literature the most common approaches to estimate breeding values are the linear or threshold statistical models. However, recent studies have shown that a Thurstonian approach was able to fix the race effect (competitive level of the horses that participate in the same race), thus suggesting a better prediction accuracy of breeding values for ranking trait. The aim of this study was to compare the predictability of linear, threshold and Thurstonian approaches for genetic evaluation of ranking in endurance horses. For this purpose, eight genetic models were used for each approach with different combinations of random effects: rider, rider–horse interaction and environmental permanent effect. All genetic models included gender, age and race as systematic effects. The database that was used contained 4065 ranking records from 966 horses and that for the pedigree contained 8733 animals (47% Arabian horses), with an estimated heritability around 0.10 for the ranking trait. The prediction ability of the models for racing performance was evaluated using a cross-validation approach. The average correlation between real and predicted performances across genetic models was around 0.25 for threshold, 0.58 for linear and 0.60 for Thurstonian approaches. Although no significant differences were found between models within approaches, the best genetic model included: the rider and rider–horse random effects for threshold, only rider and environmental permanent effects for linear approach and all random effects for Thurstonian approach. The absolute correlations of predicted breeding values among models were higher between threshold and Thurstonian: 0.90, 0.91 and 0.88 for all animals, top 20% and top 5% best animals. For rank correlations these figures were 0.85, 0.84 and 0.86. The lower values were those between linear and threshold approaches (0.65, 0.62 and 0.51). In conclusion, the Thurstonian approach is recommended for the routine genetic evaluations for ranking in endurance horses.     

Lost in translation: The search for an in vitro screen for spermatogenic toxicity

The last two decades have seen an increasing search for in vitro models that can replace the use of animals for safety testing. We adapted the methods from a recent nonquantitative report of spermatogenesis occurring in ex vivo mouse testis explants and tried to develop them into a screening assay. The model consisted of small pieces of neonatal mouse testis (testis “chunks”), explanted and placed on pillars of agarose or chamber inserts, and cultured at the air–liquid interface. A peripheral torus‐shaped zone in these explants would often contain tubules showing spermatogenesis, while the middle of each chunk was often necrotic, depending on the thickness of the tissue. The endpoint was histology: what proportion of tubules in the “permissive torus” actually contained healthy pachytene spermatocytes or spermatids? Extensive statistical modeling revealed that a useful predictive model required more than 60% of these tubules to show spermatogenesis. Separately, the logistics of running this as a predictive assay require that the controls consistently produce ≥ 60% tubules with pachytenes and round spermatids, and achieving this level of spermatogenesis reliably and consistently every week proved ultimately not possible. Extensive trials with various media additions and amendments proved incapable of maintaining the frequency of spermatogenic tubules at consistently ≥ 60%. Congruent with Schooler's “decline effect”; generally, the more often we ran these cultures, the worse the performance became. We hope that future efforts in this area may use our experience as a starting point on the way to a fully productive in vitro model of spermatogenesis.     

Evidence for a precursor for TRH in the neonatal rat pancreas

Immunoreactive TRH-OH is present at low concentrations in acid extracts from 2- days old rat pancreas. The sequential treatment of these extracts with trypsin and carboxypeptidase A is followed by a three- and ten-fold increase in TRH-OH IR respectively. The molecular weight of the protein that gives rise to TRH-OH after enzymatic treatment ranges between 30000 and 40000 daltons. The appearance of TRH-OH in the tryptic digest suggests that TRH-OH is the COOH-terminal sequence of this protein. These results are the first evidence that TRH biosynthesis occurs through a large molecule precursor. However, this is an indirect demonstration since TRH cannot be generated under these conditions due to the lack of enzymatic amidation activity.     

New evidence for dysgenic fertility for intelligence in the United States

Data were taken from the 1994 National Opinion Research Center survey of a representative sample of American adults to examine the relation between the intelligence of adults aged 40+ and their numbers of children and their numbers of siblings. The correlations were found to be significantly negative at -0.05 and -0.09, respectively, indicating the presence of dysgenic fertility. Further analysis showed that dysgenic fertility is present only in females. The correlation for females between intelligence and ideal numbers of children was effectively zero, indicating that if women had the numbers of children they consider ideal, dysgenic fertility would be reduced.     

Stereoscopic subsystems for position in depth and for motion in depth.

We describe psychophysical evidence that the human visual system contains information-processing channels for motion in depth in addition to those for position in depth. These motion-in-depth channels include some that are selectively sensitive to the relative velocities of the left and right retinal images. We propose that the visual pathway contains stereoscopic (cyclopean) motion filters that respond to only a narrow range of the directions of motion in depth. Turning to the single-neuron level we report that, in addition to neurons turned to position to depth, cat visual cortex contains neurons that emphasize information about the direction of motion at the expense of positional information. We describe psychophysical evidence for the existence of channels that are sensitive to change size, and are separate from the channels both for motion and for flicker. These changing-size channels respond independently of whether the stimulus is a bright square on a dark ground or a dark square on a bright ground. At the physiological level we report single neurons in cat visual cortex that respond selectively to increasing or to decreasing size independently of the sign of stimulus contrast. Adaptation to a changing-size stimulus produces two separable after-effects: an illusion of changing size, and an illusion of motion in depth. These after-effects have different decay time constants. We propose a psychophysical model in which changing-size filters feed a motion-in-depth stage, and suppose that the motion-in-depth after-effect is due to activity at the motion-in-depth stage, while the changing-size after-effect is due to to activity at the changing-size and more peripheral stages. The motion-in-depth after-effect can be cancelled either by a changing-size test stimulus or by relative motion of the left and right retinal images. Opposition of these two cues can also cancel the impression of motion in depth produced by the adapting stimulus. These findings link the stereoscopic (cyclopean) motion filters and the changing-size filters: both feed the same motion-in-depth stage.     

Searching for a model for use in vegetation analysis

Summary Indirect gradient analysis methods require an explicit vegetation model which must be based on direct gradient analysis studies. Various vegetation models are reviewed. Field evidence for the models is discussed. Experimental studies of species response to environmental gradients are reviewed and discussed. Three types of gradient are recognized as important for development of models: indirect environmental gradients where the environmental factor has no direct physiological influence on plant growth e.g. elevation; direct environmental gradients where the factor has a direct physiological effect on growth but is not an essential resource, e.g. pH; resource gradients where the factor is an essential resource for plant growth. The behaviour of the ecological carrying capacity and the role of competition along such gradients are shown to be important for developing vegetation models.     

Accounting for periodicities in biology

The attention of biologists is directed to the analogy between a metabolizing system and an electrical circuit with feedback and finite time delay. Periodicities can be explained on the basis of linear chemical kinetics and linear differential equations, although the latter are of infinite order.     

Evidence for a functional role for histidine in lysyl oxidase catalysis

The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.     

Role for NMR in structural genomics

The 2nd EMSL Workshop on Structural Genomics was held on 28th and 29th July 2000 at the Environmental Molecular Sciences Laboratory at the Department of Energy's Pacific Northwest National Laboratory in Richland, WA. The workshop focused on four topics: 1. The role for NMR in structural and functional genomics; 2. The technical challenges NMR faces for structural and functional genomics; 3. The potential need for a national NMR center for structural and functional genomics in the United States; and 4. Organization of the NMR community. This report summarizes the workshop proceedings and conclusions reached regarding the role of NMR in the emerging fields of structural and functional genomics.     

Radioimmunoassay for nitrazepam in plasma.

A radioimmunoassay (RIA) has been developed for the determination of therapeutic levels of the widely used hypnotic and anticonvulsant agent nitrazepam directly in 10 μl samples of plasma. The antiserum to nitrazepam, which was obtained following immunization of rabbits with an albumin conjugate of 3-hemisuccinyloxy-nitrazepam, does not cross-react with its major metabolites 7-amino-nitrazepam and 7-acetylamino-nitrazepam. The specificity of the RIA has been validated by comparison with a high-pressure liquid chromatographic procedure in the determination of intact nitrazepam in plasma following oral administration of 5 and 10 mg of the drug to man. The RIA intra- and inter-assay coefficients of variation did not exceed 7 and 9.5%, respectively. The RIA has a limit of sensitivity of 4 ng/ml using 10 μl of plasma and is ideally suited for routine clinical monitoring of nitrazepam in epileptic patients who are not receiving other benzodiazepines and for detailed pharmacokinetic and bioavailability studies in pediatric or geriatric patients from whom relatively small blood specimens are available.