[What is needed for rTMS to become a treatment?]. / Que manque-t-il à la rTMS pour devenir une thérapie?

Repetitive trans-cranial magnetic stimulation (rTMS) can modulate cortical excitability. Consequently, it appears appealing for the treatment of some affections such as depression or hallucinations. There is already some proof that the concept is valid, but rTMS is slow in progressing in the therapeutic field as a true armamentum. Indeed its effects are of short duration and even inconstant from one patient to the next. These drawbacks depend on certain factors that we will discuss. Until now, there has been inadequate control of the stimulation site. It is possible that this site could vary on an individual basis. It seems logical to propose the use of functional imaging for such a purpose, but its use should be adapted to the symptom. Even after localizing the site, the coil has to be placed accurately. This could be facilitated by a neuronavigator. Stimulation protocols are currently defined by three parameters: the frequency modulating the cortical action either as a stimulation (>5 Hz) or an inhibition (<1 Hz), the intensity and the number of stimuli influencing, notably, the amplitude and duration of the effect. Unfortunately, the effect is inconstant in a given patient and paradoxical reactions have been observed in more than 15% of normal individuals. Improved reliability and amplification of the effect rely on the better control of other parameters: pattern of stimulation, pre and post-conditioning, state of the cortex during stimulation, associated medications, endogenous idiosyncratic factors and related pathology. We will review the current physiological literature to discuss the possible options that would constitute a rational basis for setting up more efficient protocols.

Trace fear conditioning: a role for context?

Fear conditioning can be rapidly obtained over long trace intervals, but its specificity with respect to both time and stimulus is uncertain. Long-trace fear conditioning often parallels contextual conditioning, and it is sensitive to hippocampal lesions. These properties of trace conditioning are not directly addressed by timing models and multiple-time-scale models of conditioning. It is proposed that during early stages of conditioning, a joint representation of the context and the stimulus trace may underlie conditioned responses, and that discriminative processes allow the emergence of specific responses in a later stage.

Certification of the extractable contents of Cd, Cr, Cu, Ni, Pb and Zn in a freshwater sediment following a collaboratively tested and optimised three-step sequential extraction procedure.

Sequential extraction procedures have been used during the last decades to study and determine trace element mobility in aquatic sediments. However, the results obtained are operationally defined and, therefore, the significance of the analytical results is related to the extraction scheme used. So, there is a need for suitable reference materials for this type of study to enable the validation of the methodologies and to control the quality of the measurements. This paper describes the preparation of a sediment reference material, BCR-701, the homogeneity and stability studies and the analytical work performed for the certification of the extractable contents of some trace elements, following a collaboratively tested and optimised sequential (three-step) extraction procedure. The paper contains all the results and gives the methods used to certify the extractable contents (mass fractions expressed as mg kg-1 on a dry matter basis) of Cd, Cr, Cu, Ni, Pb and Zn in all steps. Moreover, indicative values are given for the aqua regia extractable contents of the six elements in the sediment and the residue obtained after sequential extraction.

Application of a modified BCR sequential extraction (three-step) procedure for the determination of extractable trace metal contents in a sewage sludge amended soil reference material (CRM 483), complemented by a three-year stability study of acetic acid and EDTA extractable metal content.

This paper provides additional data on a sewage sludge amended soil certified reference material, CRM 483, which was certified in 1997 for its EDTA and acetic acid extractable contents of some trace metals, following standardised extraction procedures. The additional work aimed to test the long-term stability of the material and the applicability of an improved version of the BCR three-step sequential extraction procedure on the sewage sludge amended soil (CRM 483). The paper demonstrates the CRM 483 long-term stability for EDTA and acetic acid extractable contents of Cd, Cr, Cu, Ni, Pb and Zn and gives the results (obtained in the framework of an interlaboratory study) for the extractable contents of the same elements in the CRM 483, following the BCR three-step sequential extraction scheme. The aqua regia extractable contents following the ISO 11466 Standard are also given. The data are given as indicative (not certified) values.

Basal body-associated DNA: in situ studies in Chlamydomonas reinhardtii.

We have explored the localization of the uni chromosome (LG XIX) of Chlamydomonas reinhardtii using the technique of in situ hybridization. Using standardized methods of cell fixation together with large chromosome-specific probes we have studied the position of uni DNA sequences in metaphase and interphase cells. We find that in dividing cells uni probes identify a condensed metaphase chromosome that shows no specialized orientation. In interphase cells uni hybridization signals occur on the anterior edge of the nucleus at a position where basal bodies are normally associated with the nuclear envelope. These data reveal an underlying spatial organization of uni chromosomal DNA within the interphase nucleus that may be significant in terms of the fact that this chromosome encodes numerous functions affecting basal body and flagellar assembly.

Single-photon emission computed tomography of the brain in acute mania and schizophrenia.

Single-photon emission computed tomography (SPECT) imaging with n-isopropyliodoamphetamine (IMP) was performed on 11 patients with bipolar mania, 21 acute schizophrenics, and 15 healthy control subjects. Subjects were evaluated with neuropsychological tests and psychiatric rating scales. SPECT brain studies were blindly evaluated to assess the degree of radiopharmaceutical uptake in three neuroanatomical regions of interest in each hemisphere. All the control subjects, 1 manic patient, and 1 schizophrenic patient had normal brain SPECT uptake patterns. The scans of all others were read as abnormal. Hypofrontality was noted in some schizophrenics and maniacs. A significant increase in tracer uptake in temporal lobes was observed in both patient groups, more prominently in the manic patients. Increased and decreased basal ganglia uptake was also observed in patients. Both manic and schizophrenic patients showed cortical tracer heterogeneity of varying degree. The patterns of cerebral SPECT uptake seen in these acute psychoses were not specific for a diagnosis, but may be associated with dimensions of psychopathology. Because the patterns are different from those seen in cerebrovascular disease and the dementias, they may prove to be helpful in differential diagnosis.

Early single-photon emission computed tomography in mild head trauma. A controlled study.

Although computed tomography (CT) and magnetic resonance imaging scans often appear normal after mild head trauma, many patients experience attentional or other cognitive disturbances that are difficult to quantify by neuropsychological testing in the absence of a premorbid profile. Within 2 days of mild head trauma, 14 patients with normal-appearing brain CTs were studied with single-photon emission computed tomography (SPECT). They were compared to 15 normal control subjects and to 12 patients with mild human immunodeficiency virus (HIV) encephalopathy. Ten of 14 head trauma patients were separated from the normal control subjects by both independent readers, blinded to the clinical diagnosis. None of the SPECT results from normal control subjects were "read" as trauma. Trauma could not be differentiated from HIV encephalopathy. The observed percentage agreement between raters was 0.83 (kappa = 0.72). SPECT is more sensitive than CT in detecting brain injury after mild head trauma.

Epitope mapping of monoclonal antibodies to bovine prolactin.

The epitopes recognized by three monoclonal antibodies generated to sheep prolactin were determined by evaluating their cross-reactivities by immunodot analysis with 14 mutants of bovine prolactin, in which individual amino acids had been deleted or substituted. Mutations were made throughout the molecule and included disruption of the amino-terminal, carboxyl-terminal, and central disulfide loops. Lack of immunoreactivity was taken as an indication that the site of mutation was part of the epitope. Antibody 6F11 reacted with all bovine prolactin mutants tested, except those in which the carboxyl-terminal cysteine (position 199) was substituted by a serine. Antibodies 5G2 and 4C10 reacted with all of the bovine prolactin mutants, except those in which the amino-terminal cysteine (position 4) was substituted by a serine. Western blot analysis of sheep, squirrel monkey, and rat prolactins with the monoclonal antibodies revealed that 5G2 and 4C10 were specific for sheep prolactin, whereas antibody 6F11 cross-reacted with prolactins from all three species. The mitogenic activity of sheep or rat prolactin in the Nb2 bioassay was determined in the presence of the antibodies to determine whether the epitopes were part of the functional domains of these prolactins. The bioactivity of sheep prolactin (0.4 ng/ml) was unaffected by the monoclonal antibodies [0.01-1 microgram immunoglobulin G (IgG)/ml], whereas the bioactivity of rat prolactin (1.25 ng/ml) was inhibited by 6F11 with an apparent 50% inhibitory concentration of 0.25 microgram IgG/ml. These results indicate that monoclonal antibodies 5G2 and 4C10 cross-react with a species-specific region of the amino-terminal disulfide loop of bovine prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of disulphide bridge function in recombinant bovine prolactin using site-specific mutagenesis and renaturation under mild alkaline conditions: a crucial role for the central disulphide bridge in the mitogenic activity of the hormone.

We have previously described a method for isolating Escherichia coli-produced methionyl bovine prolactin (Met-bPRL) and its renaturation using thioredoxin. This report describes an alternative renaturation procedure in which extracted Met-bPRL is incubated in air at pH 10 and 20 degrees C. Within 1 h of such treatment essentially all of the reduced Met-bPRL was converted to the oxidized form; this was accompanied by an increase to full mitogenic activity in the Nb2 cell bioassay. It was also found that, to minimize contamination by high mol. wt Met-bPRL derivatives, it is essential to have a reducing agent (dithiothreitol) present during disruption of the bacteria and to extract the protein at neutral pH. The contribution of each of the three disulphide bridges in bPRL to its bioactivity was studied with Met-bPRL variants, prepared via site-specific mutagenesis, in which cysteines were replaced by serines to prevent disulphide bond formation. Variants lacking the C4-C11 bridge, the C191-C199 bridge or both these terminal bridges were as mitogenic as authentic bPRL. (Variants lacking the C191-C199 bridge had markedly increased solubility in the presence of deoxycholate.) In contrast, variants lacking the C58-C174 bridge had greatly reduced bioactivity, indicating that integrity of the large disulphide loop is crucial to the hormone's mitogenic activity.

Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures.

Three amino acid residues of bovine PRL (bPRL) have been examined for their roles in the mitogenic activity of the hormone in Nb2 lymphoma cell cultures. The residues of interest, R21, R177, and K187, are conserved in eight pituitary PRLs, but not in the related, nonlactogenic bGH. Using site-specific mutagenesis, a number of recombinant methionyl bPRL variants have been prepared, each of which contained a single amino acid substitution of one of the three residues; a variety of amino acids was used for substitution. Twelve exchanges of R177 (to A, L, N, K, D, E, Y, G, S, Q, H, and F) all led to marked decreases in mitogenic activity. Even the conservative change, R177K, led to a decrease in mitogenic activity of about 90%; all the other R177 substitutions led to even more marked decreases; there was essentially complete loss of activity when the positively charged R177 was replaced by the negatively charged aspartate. Exchanges of R21 (to A, L, N, and K) were less dramatic, with the greatest decrease (79%) occurring in the case of R21A. Exchanges of K187 (to A, L, N, and R) had a relatively minor effect on the mitogenic activity of the hormone. Residues R21 and R177 in bPRL are located in putative helices 1 and 4, respectively; in the three-dimensional structure of the hormone these residues are predicted to be quite closely apposed. The results suggest that R177 and, to a lesser degree, R21 have important roles in the mitogenic activity of bPRL.

The role of SPECT brain imaging in assessing psychopathology in the medically ill.

Cerebral single photon emission computed tomography (SPECT), a method of functional brain imaging, measures cerebral blood flow and metabolism. This paper describes the imaging procedure and several cases where cerebral SPECT was of use in the differential diagnosis of medically ill patients who also presented with psychopathology. SPECT patterns in cerebrovascular disease, dementia, focal epilepsy, and AIDS are at present the best described and seem to be the most specific. Often changes in regional cerebral blood flow are seen before structural changes become apparent on CT or MRI. Cerebral SPECT can add valuable diagnostic information in assessing psychopathology in the medically ill and can often lead to changes in treatment.

Single-photon emission computed tomography in human immunodeficiency virus encephalopathy: a preliminary report.

Depression or psychosis in a previously asymptomatic individual infected with the human immunodeficiency virus (HIV) may be psychogenic, related to brain involvement by the HIV or both. Although prognosis and treatment differ depending on etiology, computed tomography (CT) and magnetic resonance imaging (MRI) are usually unrevealing in early HIV encephalopathy and therefore cannot differentiate it from psychogenic conditions. Thirty of 32 patients (94%) with HIV encephalopathy had single-photon emission computed tomography (SPECT) findings that differed from the findings in 15 patients with non-HIV psychoses and 6 controls. SPECT showed multifocal cortical and subcortical areas of hypoperfusion. In 4 cases, cognitive improvement after 6-8 weeks of zidovudine (AZT) therapy was reflected in amelioration of SPECT findings. CT remained unchanged. SPECT may be a useful technique for the evaluation of HIV encephalopathy.

Recombinant methionyl bovine prolactin: loss of bioactivity after single amino acid deletions from putative helical regions.

We have previously described a method for producing recombinant methionyl bovine PRL (Met-bPRL), which is as bioactive as the authentic hormone in the Nb2 cell lactogen bioassay; in contrast, a Met-bPRL variant lacking tyrosine 28 was essentially devoid of bioactivity. In the present study we have investigated this loss of bioactivity at the molecular level by determining the bioactivities of a number of Met-bPRL variants engineered to contain specific changes in their primary structures. It was found that the presence of tyrosine per se at the 28 position in Met-bPRL was not essential for high bioactivity, since Met-bPRL variants prepared by replacing tyrosine 28 with other amino acids (arginine, phenylalanine, alanine, and histidine) still had substantial bioactivity (40-74% that of Met-bPRL). Neither was the loss of bioactivity related to a shift in the relative positions of conserved histidines 27 and 30; in fact, histidine 27 was found not to be essential for the bioactivity of the hormone. The loss of bioactivity after deletion of tyrosine 28 from Met-bPRL appears to be related to the removal of an amino acid from the middle of a putative helix (no. 1) rather than to the loss of a residue specific to lactogen function. This suggestion is supported by the finding that Met-bPRL variants obtained by deletion of selected single amino acids from center domains of putative helix 2, 3, or 4 were also essentially devoid of bioactivity. It is speculated that this lack of bioactivity reflects an inability of the proteins to assume a native conformation.

Basal body/centriolar DNA: molecular genetic studies in Chlamydomonas.

In Chlamydomonas reinhardtii, mutations on an unusual linkage group, the uni linkage group (ULG), affect structure and function of basal bodies. The ULG shows Mendelian segregation, but its genetic map is circular. Molecular cloning of fragments of the ULG was accomplished by taking advantage of restriction fragment length polymorphisms generated by crosses to Chlamydomonas smithii. These clones were used as probes to determine the size and form of the ULG chromosome; it is a 6-9 megabase linear molecule. Use of the probes for in situ DNA hybridization in cells localized the ULG chromosome to basal bodies.

Bioactive recombinant methionyl bovine prolactin: structure-function studies using site-specific mutagenesis.

A method has been developed for the extraction from transformed Escherichia coli cells of methionyl bovine PRL (met-bPRL) in a relatively pure form. While the extracted met-bPRL was as reactive as the native hormone with respect to polyclonal anti-bPRL antibodies, its bioactivity, as measured by the Nb2 lactogen in vitro bioassay, was relatively low. The bioactivity of the met-bPRL could be increased to the same order as that of the native hormone by treatment with a mixture of oxidized and reduced thioredoxin. A number of variant met-bPRLs containing specific amino acid changes have been generated by site-specific mutagenesis. The changes involved the substitution (or deletion) of some of the conserved amino acids in bPRL by the different amino acids present at the corresponding positions in the related, but nonlactogenic bovine GH. Nine mutants containing single amino acid changes had bio- and immunoactivities of the same order as those of met-bPRL. One mutant, which incorporated two of the single amino acid changes (serine 62 to threonine and threonine 65 to alanine), had immunoactivity approximating that of met-bPRL but much lower bioactivity (45%). A further mutant, generated by the deletion of tyrosine 28, had essentially no bioactivity although it could not be distinguished immunologically from met-bPRL or bPRL. The findings are discussed in the light of the putative three-dimensional PRL structure and current hypotheses which seek to relate specific regions of PRL to lactogenic activity. It appears that the first putative alpha-helix of bPRL is important for the binding and mitogenic activity of the hormone.

Single photon emission computed tomography (SPECT) with [123I]IMP in the differential diagnosis of psychiatric disorders.

Single photon emission computed tomography (SPECT) with [123I]N-isopropyl iodoamphetamine--[123I]IMP--was used to study regional cerebral blood flow (rCBF) in 94 psychiatric patients and six controls. Patients with dementias had distinct brain-image patterns, consistent with the expected neuropathology. Major depressives had decreased cortical and subcortical rCBF. Increased caudate rCBF was observed in schizophrenics and other psychoses with positive symptoms. SPECT is a practical method of functional brain imaging with potential in the differential diagnosis of neuropsychiatric disorders. Further studies are needed to assess the effect of other variables on brain-image patterns.

Synthesis of bovine prolactin in Escherichia coli.

Transformation of Escherichia coli cells with a recombinant plasmid (pESP4) containing a modified bovine prolactin cDNA clone in a pEMBL vector resulted in efficient expression of prolactin. The cDNA was modified by removal of a 5' nontranslated sequence as well as the sequence that specified the signal peptide of preprolactin. To achieve a high level of synthesis, a sequence of 30 nucleotides in the cDNA, which included the ATG initiation codon and the first 7 codons of mature bovine prolactin, was replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen from the consensus sequence around the initiation codon of E. coli genes and by the amino acid sequence of the protein. Prolactin, a single-chain polypeptide of molecular weight 24,000, was identified by Coomassie Blue staining of NaDodSO4-polyacrylamide gels of total protein from transformed E. coli cells, and by reaction with specific antibody. Increased levels of expression of the hormone, corresponding to the form secreted from the pituitary, were observed in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG).

Loci affecting flagellar assembly and function map to an unusual linkage group in Chlamydomonas reinhardtii.

The uni1 mutant of Chlamydomonas reinhardtii lacks one of the paired flagella seen in wild-type cells. The missing flagellum is cis to the eye spot and at this site the basal body is incomplete, lacking a transition zone. Together with nine other loci affecting flagellar assembly and two loci affecting flagellar function uni1 defines an approximately equal to 100-centimorgan linkage group with a circular map. Measurements of gene-centromere distances on the uni linkage group are consistent with the ordering of loci determined by recombination analysis and place the centromere near pf29, a motility-defective mutant. In well-synchronized meiotic cycles, recombination frequencies between loci on the uni linkage group but not loci on other linkage groups show striking temperature sensitivity during a 1- to 2-hr period 5 days before meiosis.

Phosphorylation in isolated Chlamydomonas axonemes: a phosphoprotein may mediate the Ca2+-dependent photophobic response.

An in vitro system was devised for studying phosphorylation of Chlamydomonas reinhardtii axonemal proteins. Many of the polypeptides phosphorylated in this system could be identified as previously described axonemal components that are phosphorylated in vivo. The in vitro system apparently preserved the activities of diverse axonemal kinases without greatly altering the substrate specificity of the enzymes. The in vitro system was used to study the effect of calcium concentration on axonemal protein phosphorylation. Calcium has previously been demonstrated to initiate the axonemal reversal reaction of the photophobic response; the in vitro system made it possible to investigate the possibility that this calcium effect is mediated by protein phosphorylation. Calcium specifically altered the phosphorylation of only two axonemal proteins; the phosphorylation of an otherwise unidentified 85,000 Mr protein was repressed by calcium concentrations greater than or equal to 10(-6) M, while the phosphorylation of the previously identified 95,000 Mr protein b4 was stimulated by calcium at concentrations greater than 10(-6) M. Protein b4 is one of six polypeptides that are deficient in the mbo mutants, strains that do not exhibit a photophobic reversal reaction. Therefore, this calcium-stimulated phosphorylation may be involved in initiating the photophobic response. Neither calmodulin nor the C-kinase could be implicated in b4 phosphorylation. The calcium-dependent activation of the b4 kinase was not affected by several drugs that bind to and inhibit calmodulin, or by the addition of exogenous calmodulin. Activators and inhibitors of the calcium-phospholipid-dependent C kinase also had no effect on b4 phosphorylation.

Bending patterns of chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant.

Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the "wild-type" uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.