Homogeneous assay based on 52 primer sets to scan for mutations of the ABCA1 gene and its application in genetic analysis of a new patient with familial high-density lipoprotein deficiency syndrome

We have applied the transition state theory of Eyring et al. (The Theory of Rate Processes, McGraw-Hill, 1941) to water transport across cell membranes. We have then evaluated free energy (Delta F(not equal)), enthalpy (Delta H(not equal)) and entropy (Delta S(not equal)) of activation for water permeation across membranes, such as Arbacia eggs, Xenopus oocytes with or without aquaporin water channels, mammalian erythrocytes, aquaporin proteoliposomes, liposomes and collodion membrane. Delta H(not equal) was found to be correlated with Delta S(not equal). This is so-called Delta H(not equal) and Delta S(not equal) compensation over the ranges of Delta H(not equal) and Delta S(not equal) from 2 to 22 kcal/mol and from -26 to 45 e.u., respectively, indicating that low Delta H(not equal) values correspond to negative Delta S(not equal). Large positive Delta S(not equal) and high Delta H(not equal) values might be accompanied by reversible breakage of secondary bonds in the membrane, presumably in membrane lipid bilayer. Largely negative Delta S(not equal) and low Delta H(not equal) values for aquaporin water channels, aquaporin proteoliposomes and porous collodion membrane could be explained by the immobilization of permeating water molecules in the membrane, i.e., the partial loss of rotational and/or translational freedoms of water molecules in water channels.     

Biofeedback efficacy studies

Biofeedback, a field still in its infancy, has developed treatments that have been used with clinical success in the treatment of a number of disorders. Many have expressed their public concern that biofeedback has not lived up to its early promise and that it has not developed treatments that are, in fact, efficacious. A number of factors, which are inherent in biofeedback research, confound the results of clinical efficacy studies of biofeedback treatments. Researchers interested in the efficacy of biofeedback must address several issues: (1) Rejecting the null hypothesis is not equal to proving the null hypothesis (without the use of power analysis); (2) control for nonspecific effects is not equal to a double-blind experimental design; (3) ignorance of a mechanism of action is not equal to a lack of clinical efficacy; (4) the administration of training is not equal to the subject's learning to criterion; (5) untrained therapists are not equal to trained therapists; (6) statistical significance is not equal to clinical significance; and (7) the laboratory setting is not equal to the clinical setting.     

Effect of methadone administration on MHV-3 virus-induced experimental hepatitis in the mouse

Mice treated with 15 mg/Kg/day methadone and infected with MHV-3 virus after 7 days did not show increased susceptibility to MHV-3 virus infection, did not develop more serious forms of hepatitis and not mortality did not increase with respect to the controls. Drug administration was continued for the duration of the experiment.     

Ten false ideas about New Caledonia biogeography

The biogeographical paradigm of New Caledonia has recently changed. Although this island is now considered by many as oceanic, its study is still often impeded by some old misconceptions concerning either regional geology or phylogenetic analysis of evolution and biogeography. I discuss ten points that I feel are especially detrimental, to help focus on the real debate and the real questions: (1) its geological history cannot be understood from the basement only; (2) the island submergence was not due simply to sea‐level variation; (3) Zealandia/Tasmantis is not a lost continent; (4) short‐distance dispersal is not equivalent to permanence on land; (5) long‐distance dispersal is not the sole event opposing vicariance, but short‐distance dispersal as well; (6) the occurrence of relicts does not prove biota permanence; (7) a major fault system was not observed in New Caledonia; (8) terranes are not rafts; (9) forest climatic refuges do not necessarily equate to centres of endemism or centres of diversity; and (10) New Caledonia is not only a sink but also a source. Study of New Caledonia will need to focus on old and non‐relict clades and there is a need to improve the local fossil record.     

A long view of fashions in cancer research

Despite the spectacular contributions to knowledge made by molecular biology during the last half century, cancer research has not delivered an agreed explanation of how malignant tumours originate. The models assiduously investigated in molecular terms largely reflect waves of fashion, and time has revealed their inadequacy: cancer is (1) not caused by the direct action of oncogenes, (2) not fully explained by the impairment of tumour suppressor genes, (3) not set in motion by mutations controlling the cell cycle, (4) not governed by the dependence of malignant tumours on an adequate blood supply and (5) not triggered by a failure of programmed cell death. But there is now strong evidence that cancers may have their origin in mutations that block the execution of critical steps in the process of normal differentiation. Cancer, thus seen, is not initially a disease of cell multiplication, but a disease of differentiation. The evidence for this point of view should now be explored.     

Building multiple nests is associated with reduced breeding performance in a south temperate population of Grass Wrens Cistothorus platensis platensis

Grass Wrens Cistothorus platensis build two types of non-breeding nest structures: platforms and dummy nests. Platforms are rudimentary accumulations of grasses concealed between vegetation. Dummy and breeding nests are dome-shaped with a similar structural layer. We used a nest-removal experiment and observational data to evaluate several hypotheses regarding the adaptive significance of building multiple nests in a south temperate population of Grass Wrens. Building non-breeding nests was not a strategy of males to attract additional females, as most of these nests were built after pair formation and both sexes collaborated during building. Building non-breeding nests was not a post-pairing display as the presence of multiple nests did not increase female investment in the breeding attempt: clutch size and female provisioning to nestlings did not differ between experimental and control territories where no non-breeding nests were removed. Similarly, in non-manipulated territories, clutch size and female provisioning were not correlated with the number of non-breeding nests or with males’ nest-building effort. Contrary to this hypothesis, the number of non-breeding nests was associated with delayed clutch initiation and reduced hatching success. The presence of non-breeding nests did not reduce nest predation and brood parasitism, which did not differ between experimental and control territories. We did not detect differences in concealment between non-breeding and breeding nests, suggesting that non-breeding nests were not the result of abandonment before egg-laying to reduce subsequent nest predation. Dummy nests did not provide shelter; they were not used frequently for roosting over the breeding season and were not maintained during the non-breeding season. We suggest that building non-breeding nests may be an attempt by males to manipulate the decision of females to breed with a mate they might otherwise reject or to start reproduction earlier than optimal for the females.     

Studies on the nature of protease-induced growth stimulation in normal and transformed BHK cells.

In the presence of growth-limiting serum concentrations trypsin displays mitogenic activity on actively-growing but not quiescent BHK cells. These results suggest that BHK cells arrested in G1 (G0) are not sensitive to protease-induced growth stimulation. Previous work strongly suggested that the trypsin active-site is not directly involved in its mitogenic activity on BHK cells. Additional studies on denatured trypsin fragments further indicate that the molecular conformation and size of native trypsin may not be absolutely required for mitogenic activity. Cellular multiplication induced by the addition of fresh serum to quiescent BHK cultures is not inhibited by high concentrations of soybean trypsin inhibitor. Similar to our previous findings with trypsin, it has been further observed that plasmin is not sufficient to initiate the growth of BHK cells in soft agar. Trypsin also fails to enhance the growth of a thermosensitive polyoma-transformed BHK line in soft agar at the restrictive temperature. Finally, the growth of transformed BHK cells in soft agar does not display a requirement for plasminogen and is not inhibited by soybean trypsin inhibitor. These studies argue against the involvement of plasmin or other exogenous trypsin-like enzymes in the growth and transformation of BHK cells.     

Rules for the use of model organisms in anti-aging pharmacology

The use of animal models for initially screening anti-aging drugs is a promising approach for drug discovery. However, there a number of potential artifacts, confounds and errors that can arise in such research programs. The following rules are intended to minimize such problems: (1) since aging occupies an increasing proportion of human adulthood, data that conflate aging and late life should not be extrapolated to human aging; (2) the response to candidate medications should show a normal dose-response pattern, although not necessarily a linear response; (3) medicated animal models should not be hypometabolic; (4) medicated animal models should not show pronounced reductions in fertility; (5) medicated animal models should not exhibit general nervous system depression; (6) the effect of the medication should not be highly sensitive to the culture environment; (7) the effect of the medication should not be highly dependent on the genetic ancestry of the stock employed, leaving aside inbreeding, which should be avoided because humans are not generally inbred. While these rules do not guarantee successful extrapolation of successful drug results from the animal model to humans in a clinical setting, the failure to adhere to these rules should raise doubts about such extrapolation.     

Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats: assessment of potential neurotrophic effects

The potential functional significance of nerve growth factor (NGF) receptors in spinal motoneurons was studied in newborn rats. 125I-NGF was specifically retrogradely transported by motoneurons from their peripheral nerve terminals. This transport was blocked by an excess of unlabeled NGF but not by cytochrome c. 125I-cytochrome c was not transported. The monoclonal anti-rat NGF receptor antibody, but not a control antibody, was also transported. Despite this ability of motoneurons to transport NGF, treatment of newborn rats with this factor did not increase motoneuron size or synthesis of neurotransmitter enzymes and did not prevent cell death after axotomy. We conclude that NGF receptors of spinal motoneurons can bind, internalize, and retrogradely transport NGF. However, these receptors do not mediate the classic trophic effects of NGF.     

Circadian variations of haemolymph lipoprotein of Palaemon serratus

The electrophoretic behaviour of haemolymph lipoprotein was determined throughout a 24 h light-dark cycle. All of the 14 lipoprotein bands did not always appear at the same time, and their level did not remain constant, showing mainly tri- and tetracircadian variations. Values are not the same in males and females. Despite the fact that the lipoprotein variation was not closely synchronized with circadian periodicity, some lipoproteins were responsible for light-dark transition.     

The Median Test and Inhomogeneity of Slopes

By some examples it is shown that the median test is not robust against deviations from homogeneity of slopes. It is also shown that it is not conservative if this assumption does not hold true.     

Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans

We have examined the effects of the antimicrotubule agent benomyl and several mutations on nuclear and mitochondrial movement in germlings of the filamentous fungus Aspergillus nidulans. While, as previously reported, benomyl inhibited nuclear division and movement, it did not inhibit mitochondrial movement. To test the effects of benomyl more rigorously, we germinated two benomyl super-sensitive, beta-tubulin mutants at a benomyl concentration 50-100 times greater than that required to inhibit colony formation completely. Again nuclear division and movement were inhibited, but mitochondrial movement was not. We also examined conditionally lethal beta-tubulin mutations that disrupt microtubule function under restrictive conditions. Nuclear division and movement were inhibited but, again, mitochondrial movement was not. Finally we examined the effects of five heat-sensitive mutations that inhibit nuclear movement but not nuclear division at restrictive temperatures. These mutations strongly inhibited nuclear movement at a restrictive temperature but did not inhibit mitochondrial movement. These data demonstrate that the mechanisms of nuclear and mitochondrial movement in Aspergillus nidulans are not identical and suggest that mitochondrial movement does not require functional microtubules.     

Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved sites.     

Effects of penitrem A on rat's performances in passive avoidance and Morris water maze tests

Intraperitoneal administration of the mycotoxin penitrem A 30 min before a training session in passive avoidance task, impaired performance of rats subjected to a test-session 24 h after. This effect was not antagonised by pretraining administration of physostigmine or bicuculline. Administration of penitrem A 20 min before a training session or 30 min before a test-session did not impair performance. In the Morris water maze, doses of penitrem A that induces slight to moderate tremors, but not a lower dose, disrupted place learning. These results suggest that penitrem A disrupts the processes that take place at the time of acquisition, but not those just after acquisition, and does not alter the restitution of information. This effect would not be related to a decrease of cholinergic neurotransmission nor to a stimulation of GABA A receptors. Nevertheless, it could not be totally excluded that the performance impairments induced by penitrem A would be secondary to a motor disruption. This revised version was published online in June 2006 with corrections to the Cover Date.     

On the Applicability of Multidimensional Classification Procedures

An example from the EEG-analysis shows that particularly in using the R-error estimation the variance analysis and discrimination might indicate contradictory results. Therefore the use of results of MANOVA for a subsequent discrimination appears not to be advisable as a rule. If the number of test persons per class does not amount to at least the five-fold of the number of characters, the error estimation according to the R-method is not applicable. Since the feature selection by the methods investigated in this paper only pretend an improvement of the classification, producing even misleading results in single cases, it should not be used, at least not with smaller random samples.     

Energy metabolism of spermatozoa. IV. Effect of calcium on respiration of mature epididymal sperm of the rabbit.

The effect of calcium (Ca+2) on the respiration rate of mature rab bit epididymal sperm was studied. The addition of Ca+2 did not further stimulate the respiration rate of sperm already stimulated by glucose or pyruvate. Oligomycin, which inhibits mitochondrial ATP synthesis and slows respiration, did not inhibit the uptake of mitochond rial Ca+2. The addition of the ionophore A23187, which promotes selective permeability of cell membranes to Ca+2, caused a marked stimulation of respiration when Ca+2 was added, indicating that the sperm cell membrane is not permeable to Ca+2. The stimulation of the respiration rate by pyruvate, but not glucose, was enhanced by the addition of 45 mM HCO3, which did not affect the response to added Ca+2. With or without Ca+2, cyclic AMP and dibutyl cyclic AMP did not stimulate respiration in the presence of pyruvate or glucose. The results suggest that mature rabbit sperm from the cauda epididymis are intrinsically motile, and not dependent on Ca+2.     

Differential effect of monoclonal anti-DR antibody on monocytes in antigen- and mitogen-stimulated responses: Mechanism of inhibition and relationship to interleukin 1 secretion

A differential role for DR antigens on monocytes in antigen-stimulated as opposed to mitogen-stimulated human lymphocyte responses has been observed. A monoclonal anti-DR antibody used to treat monocytes caused inhibition of antigen-induced T-cell responses and of T-cell-dependent B-cell responses. However, anti-DR antibody treatment of monocytes did not inhibit mitogen-induced responses. Anti-DR treatment of monocytes did not induce suppression, as antigen-induced responses could be reconstituted with untreated monocytes. Anti-DR treatment of monocytes did not merely block interleukin 1 (IL-1) secretion since addition of IL-1 could not restore antigen-induced responses. Monoclonal anti-DR antibody did not directly inhibit monocyte secretion of IL-1. DR-negative monocytes, selected by antibody and complement, could not present antigen, even though they were capable of secreting IL-1. Thus, this monoclonal anti-DR antibody sterically blocks antigen presentation by monocytes without induction of suppression or inhibition of IL-1 secretion. Monocyte DR antigens appear essential for stimulation of antigen-induced responses, but DR antigens on monocytes may not be essential for mitogen-stimulated responses and do not appear to be related to the ability of monocytes to secrete IL-1.     

6-Hydroxydopamine produces a weight increase in toads

A large increase in body weight occurred after injection of 6-hydroxydopamine (6-OHDA, 100 mg kg-1) into the iliac lymph sac of toads. The body weight increase was apparently due to retention of fluid and only occurred when toads could not avoid contact of their ventral skin with water. Lower doses of 6-OHDA (25 or 50 mg kg-1) did not cause a weight increase although they were sufficient to eliminate fluorescence from peripheral adrenergic nerves. A peripheral injection of 6-OHDA did not affect the appearance of central aminergic nerves. Removal of the pituitary and median eminence did not affect the weight increase. Although the primary action of 6-OHDA in upsetting water balance in toads has not been revealed by this study, it is clearly not related to the chemical sympathectomy of adrenergic nerves.     

Leptin does not directly affect CNS serotonin neurons to influence appetite

Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sert(cre)) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sert(cre) promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sert(cre)-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function.