Diet: the keystone of autism spectrum disorder?

Children with autism are characterized by an impairment of social interaction and repetitive patterns of behaviour. Autism is a heterogeneous span of disorders with unknown aetiology. Research has grown significantly and has suggested that environmental risk factors acting during the prenatal period could influence the neurodevelopment of offspring. The literature suggests that the maternal diet during pregnancy has a fundamental role in the etiopathogenesis of autism. Indeed, a maternal diet that is high in some nutrients has been associated with an increase or reduction in the risk of develop Autism Spectrum Disorders (ASD). The diet of ASD children is also a key factor for the worsening of ASD symptoms. Children with autism have food selectivity and limited diets due to smell, taste, or other characteristics of foods. This determines eating routines and food intake patterns, with consequent deficiency or excess of some aliments. Several studies have tried to show a possible relationship between nutritional status and autism. In this review we describe, emphasizing the limits and benefits, the main current empirical studies that have examined the role of maternal diet during gestation and diet of ASD children as modifiable risk factors at the base of development or worsening of symptoms of autism.

Safety and tolerability considerations: tricyclic antidepressants vs. selective serotonin reuptake inhibitors.

OBJECTIVE: An important consideration in the choice of an antidepressant is its safety and tolerability. METHOD: We present a review of literature, clinical trials and meta-analyses regarding the safety and tolerability of the tricyclic antidepressants (TCAs) and the selective serotonin reuptake inhibitors (SSRIs) in depressed patients. RESULTS: The SSRIs have a very favourable side-effect profile compared to the TCAs and are associated with fewer treatment discontinuations. Unlike the TCAs, they do not cause anticholinergic, hypotensive or sedating reactions, and are not associated with impaired cognitive function. Their most common side-effects (nausea, vomiting, nervousness, insomnia, headache and sexual dysfunction) are usually mild and typically disappear as treatment continues. The SSRIs also exhibit lower toxicity and lower lethality when taken in an overdose situation. Although the safety profiles of the principal SSRIs appear to be comparable, there is some data showing important differences in the severity and frequency of specific adverse events. CONCLUSION: The SSRIs have a more favourable safety profile than the TCAs in both acute and long-term treatment of major depression.

Coordinated clearance of periciliary liquid and mucus from airway surfaces.

Airway surface liquid is comprised of mucus and an underlying, watery periciliary liquid (PCL). In contrast to the well-described axial transport of mucus along airway surfaces via ciliary action, theoretical analyses predict that the PCL is nearly stationary. Conventional and confocal microscopy of fluorescent microspheres and photoactivated fluorescent dyes were used with well-differentiated human tracheobronchial epithelial cell cultures exhibiting spontaneous, radial mucociliary transport to study the movements of mucus and PCL. These studies showed that the entire PCL is transported at approximately the same rate as mucus, 39.2+/-4.7 and 39.8+/-4.2 micrometer/sec, respectively. Removing the mucus layer reduced PCL transport by > 80%, to 4.8+/-0.6 micrometer/sec, a value close to that predicted from theoretical analyses of the ciliary beat cycle. Hence, the rapid movement of PCL is dependent upon the transport of mucus. Mucus-dependent PCL transport was spatially uniform and exceeded the rate expected for pure frictional coupling with the overlying mucus layer; hence, ciliary mixing most likely accelerates the diffusion of momentum from mucus into the PCL. The cephalad movement of PCL along airway epithelial surfaces makes this mucus-driven transport an important component of salt and water physiology in the lung in health and disease.

The state of functioning of working memory in schizophrenia.

The functioning of working memory in schizophrenic patients according to Baddeley's model was examined in two complementary experiments. Experiment 1 comprised 27 patients and their controls, matched in age and level of education. Of this pool, 20 pairs participated also in Experiment 2. Digit span, reading rate, and immediate serial recall assessed the functioning of the phonological loop. Corsi and pattern span tasks assessed the capacity of visuo-spatial memory. The central executive's ability to monitor two concurrent tasks was evaluated in a dual task paradigm, and its capacity to control action in a random generation task. A preliminary set of analyses showed that the patients' performances were reduced in all tasks explored, except in digit span. This initial pattern changed consistently after controlling for reading rate. While slow and fast reading patients were comparable in demographic and clinical criteria, slow reading patients showed impaired performance in all tasks, whereas fast reading patients exhibited reduced performance in visuo-spatial tasks and in the random generation task only. The state of functioning of working memory in schizophrenia appears, therefore, to vary consistently among the components of the model and is markedly impaired in slow reading patients. The implications of slowness are discussed.

Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. I. Experiments.

A strain of Pseudomonas putida harboring plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor under three different concentrations of the limiting nutrient, succinate. Experimental results demonstrated that the broad host range RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. At the lower concentrations, donor mediated plasmid transfer increased with increasing nutrient levels, but the highest nutrient concentration yielded the lowest rate of donor to recipient plasmid transfer. For transconjugant initiated transfer, the rate of transfer increased with increasing nutrient concentrations for all cases. At the lower nutrient concentrations, the frequency of plasmid transfer was higher between donors and recipients than between transconjugants and recipients. The reverse was true at the highest succinate concentration. The rates and frequencies of plasmid transfer by mobilization were compared to gene exchange by retrotransfer. The initial rate of retrotransfer was slower than mobilization, but then increased dramatically. Retrotransfer produced a plasmid transfer frequency more than an order of magnitude higher than simple mobilization.

Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. II. Modeling.

A strain of Pseudomonas putida that harbors plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor. Transfer of the RK2 mobilizable pDLB101 plasmid to B. azotoformans was monitored over a 4-day period. Experimental results demonstrated that the broad host range, RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. In the companion article to this work, the rate of plasmid transfer was quantified as a function of the limiting nutrient, succinate, and as a function of the mechanism of transfer. A biofilm process simulation program (AQUASIM) was modified to analyze resultant experimental data. Although the AQUASIM package was not designed to simulate or predict genetic events in biofilms, modification of the rate process dynamics allowed successful modeling of plasmid transfer. For the narrow range of substrate concentrations used in these experiments, nutrient level had only a slight effect on the rate and extent of plasmid transfer in biofilms. However, further simulations using AQUASIM revealed that under nutrient poor conditions, the number of transconjugants appearing in the biofilm was limited.

Continuous culture dynamics for aniline metabolism by Pseudomonas sp. CIT1.

Inhibition by toxic substrates enables multiple steady states to arise in biodegradation systems. This phenomenon was investigated for the continuous metabolism of aniline by Pseudomonas sp. CIT1. Differences of various metabolic parameters between the two growth regimes (uninhibited and inhibited) and the transient response to a step-up in dilution rate were determined. Regulatory mechanisms consistent with the experimental evidence are proposed. Aniline is the transcriptional inducer of a metabolic pathway that converts aniline to TCA cycle intermediates. The suite of enzymes is coordinately expressed from a single promoter. We followed the level of the pathway mRNA using a fragment containing the catechol 2,3 dioxygenase gene (andioxB) and monitored the pathway enzyme activity using catechol 2,3 dioxygenase (C23D). The inhibited regime resulted in a 60% lower growth yield, near constant levels of C23D monomer, but a 50% reduction in the specific activity of C23D, increased RNA synthesis rates (total and aniline pathway mRNA), and elevated RNA decay rates. Elucidation of regulatory mechanisms indicates that C23D is noncompetitively inhibited by aniline and subject to feedback inhibition by 2-hydroxymuconic semialdehyde (HMS). During uninhibited growth regime operation, metabolism of HMS is the rate-limiting step; in contrast, conversion of aniline to catechol limits growth in the inhibited regime.

[Results of specific neonatal screening for congenital dysplasia of the hip at the Vicenza Hospital. A prospective study of subjects at risk]. / Risultati di uno screening mirato per displasia congenita dell'anca, in neonati presso l'ospedale civile di Vincenza. Studio prospettico in soggetti a rischio.

The aim of the study was to evaluate the incidence of pathological neonatal hips in the community served by the central Vicenza local health authority and to establish a protocol to minimize demands on available equipment, staff and the family. From May 1992 to May 1993 all neonates at San Bortolo Hospital in Vicenza were subjected to specific clinical examination of the hip by staff experienced in neonatal care. Neonates then underwent ultrasound examination--catalogued according to Graf--if they presented risk factors (dynamic ultrasound test was omitted). The orthopaedic examination was carried out in all cases. The total number of neonates involved was 1939 (994 m., 945 f.). Of these, 142 (7.3%) underwent ultrasonography (60 m., 82 f.). Family history and breech delivery were the most frequent anamnestic risk factors justifying ultrasound examination while among objective risk factors the most frequent being a clicking sound. Considering the clinical and ultrasound findings the resulting overall incidence of pathological hips is 0.25%. So far, there have been no late cases of c.d.h. A screening protocol such as ours cannot realistically aim to identify all pathological hips, however the great majority can be diagnosed at this early stage. Resources comparable to those used for our study are available to many other local health authorities. Higher diagnostic standard depend closely on local health policy.

Prenatal stress attenuates ulceration in the activity stress paradigm.

In the present study, 28 pregnant rats were subjected to either light-restraint stress or no manipulation for days 14-21 of the gestational period. At approx. 50 days of age, both male (n = 16) and female (n = 16) prenatally stressed (PS) and control offspring were subjected to the activity stress (AS) paradigm. During this subsequent stress experience, PS rats developed less ulceration than control rats. PS rats also displayed about half the activity of the control animals during the habituation phase of the AS paradigm, prior to the induction of stress. Given this decrease in baseline activity in PS animals, implications of using activity as a measure of emotionality in PS animals are discussed. Several sex differences were also observed; females differed from males in that they 1) exhibited higher activity levels in both the habituation and experimental phases of the AS procedure, 2) developed heavier relative adrenal weights, and 3) reached criteria for sacrifice in fewer days.

Effects of medium carbon-to-nitrogen ratio on biofilm formation and plasmid stability.

Biofilm formation and plasmid segregational instability in biofilm cultures of Escherichia coli DH5alpha (pMJR1750) were investigated under different medium-carbon-to-nitrogen (C/N) ratios. At C/N ratios of 0.07 and 1, net accumulation of both biofilm plasmid-bearing and plasmid-free cells continued through the entire experiment without attaining any apparent steady state. At C/N ratios of 5 and 10, net biofilm cell accumulation for the two populations reached apparent steady states after 84 and 72 h, respectively. At C/N ratios of 0.07 and 1, polysaccharide production increased slowly and reached about 2g alginate equivalent/cm(2) by the end of both experiments. At a C/N ratio of 5, polysaccharide increase significantly after 84 h, reaching about 7 microg alginate equivalent/cm(2) prior to termination. At a C/N ratio of 10, polysaccharide increased significantly after 72 h and reached 21 microg alginate equivalent/cm(2) at 108 h. At C/N ratios of 0.07 and 1, protein production reached 6.5 and 4 microg/cm(2), respectively. At C/N ratios of 5 and 10, protein production increased slightly for the first 84 h and reached a maximum at 108 h, at 3 and 2 microg/cm(2), respectively, then decreased over the last 12 h of the experiment. Ratios of polysaccharide to protein increased with increasing C/N ratios. At C/N ratios of 0.07 and 1, the ratios between extracellular polysaccharide (EP) and protein were no more than 205 microg polysaccharide/microg protein, whereas those at C/N ratios of 5 and 10 increased to about 7 and 12 microg polysaccharide/microg protein, respectively.Probabilities of plasmid loss in the biofilm cultures increased with increasing C/N ratios. At C/N ratios of 0.07, 1, and 5, the probabilities of plasmid loss were 0.0013 +/- 0.011, 0.020 +/- 0.006 and 0.122 +/- 0.021, respectively. At a C/N ratio of 10, the probability of plasmid loss was significantly higher, reaching 0.38 +/- 0.125. The increase of probability of plasmid loss at higher C/N ratios results from competition between cell replication and extracellular polysaccharide production.

Stimulation of berberine secretion and growth in cell cultures of Thalictrum minus.

An endo-pectate lyase (PL; EC 4.2.2.2), originally cloned fiom the phytopathogenic bacterium Erwinia chrysanthemi EC16, was expressed in recA (-) E. coli strain DK1, purified to a single band by isoelectric focusing and used to induce berberine production in established plant suspension cultures of Thalictrum minus L. subsp. saxatile. Addition of 10(-9)M pectate lyase c (PLc) stimulated berberine production and enhanced secretion of the alkaloid into the medium. A lower concentration of PLc, 10(-11)M, stimulated a transient two-fold increase in cell growth rate relative to untreated cultures. Parallel changes in L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity with the rate of berberine synthesis and the inverse relationship between cell growth and berberine synthesis imply that berberine synthesis is stress-related in this cell line.

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750).

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively.

Analysis of productivity in lysis-deficient lambda expression systems.

The integrated state of lambda in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on lambda expression systems(21,22) have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of lambda systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential lambda protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector lambdaZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in lambda systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of lambda vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems.

[Memory disorders in schizophrenia]. / Les troubles de la mémoire dans la schizophrénie.

The current interest in memory disorders in schizophrenia results from the way perceptions of schizophrenia--whose organic origin is becoming increasingly evident--and memory--according to which there exist not one, but several memories--have developed. Memory disorders in the schizophrenic cannot be considered in isolation from knowledge accumulated in other areas of the cognitive and neuro-sciences; a more detailed understanding of these disorders requires a comparison of the different cognitive approaches, both with each other and with the neurobiological and clinical approaches, so that they can be integrated. Despite numerous methodological and conceptual difficulties, it now appears to have been established that the schizophrenic's memory deficit should be seen in the context of a wider cognitive deficit, that the memory tasks are not all disturbed and that the memory deficit cannot be identified with one specific form of memory. Thus, iconic formation, short-term memory in the traditionally accepted sense and implicit memory are hardly, if at all, affected; in contrast, the early processing of information, working memory and explicit memory are disturbed, probably to the extent that they require the implementation of strategies to organise the information to be memorized. Finally, in certain tasks, such as those evaluating latent inhibition or negative priming, schizophrenics perform better than normal subjects, suggesting that schizophrenics' cognitive deficit is localised. This profile of memory disorders is compatible with a dysfunction predominating in the frontal and temporo-hippocampal regions. Neuroleptics and anticholinergics have opposite effects on cognitive and mnesic performance, which is improved by the former and aggravated by the latter. The influence of clinical symptoms, positive or negative, institutionalisation of patients and chronic tardive dyskinesia is unclear. Among the theoretical proposals put forward to account for the observed disorders, those relating to a disturbance of the action planning process and to that of the internal representation of context are compatible with the observed memory disorders. All the clinically derived data and those produced by the cognitive and neurosciences indicate a need to reformulate the links between memory, selective attention and evaluation of the relevance of a stimulus, to develop a general model of the reciprocal interactions between cognition and affectivity and to look for the origin of a pathology as complex as schizophrenia, not in a local lesion in an isolated cerebral structure but in a disturbance of the dynamic interactions within a functional, parallel and distributed network of broadly interconnected regions.

Characterization of the mutant lytic state in lambda expression systems.

The two propagative phases of bacteriophage lambda, lysogeny and lysis, can be used in concert to enhance productivity of recombinant expression systems. Lambda vectors carrying mutations to prevent both cell lysis and lambda DNA packaging in the lytic state have been shown to yield 100% stability of the product gene in lysogeny and to produce up to 15% of total cell protein as product beta-galactosidase in a mutant lytic state.(14) Despite these mutations, partial lysis of the culture was observed following induction of the cells from a lysogenic phase into the lytic state. To understand better the phage-host cell interactions and to investigate the possible cause(s) of lysis in these highly productive expression systems, we have made a detailed study of the suppressor-free system JM105(NM1070). We have found high levels of product (15% of total cell protein as beta-glactosidase) to be due chiefly to a high-copy number of lambda DNA in the mutant lytic state. There is partial lysis of the culture even in this suppressor-free system caused by a low-level natural suppression of the amber mutation in gene S of NM1070, resulting in accumulation of lambda endolysin. We have also monitored changes in cell growth and morphology upon induction of the lysogen. There is a slight increase in cell number that follows a linear relationship with time and a 25-fold increase in cell volume during recombinat protein production in the mutant lytic state.

Effects of chlorpromazine and lorazepam on explicit memory, repetition priming and cognitive skill learning in healthy volunteers.

To assess the influence of neuroleptics on explicit memory and two forms of implicit memory, repetition priming and cognitive skill learning, the effects of two low doses of chlorpromazine (12.5 and 25 mg orally) were contrasted to those of lorazepam (2.5 mg orally) and of a placebo using a free-recall task, a word-completion task and repeated testing on the Tower of Toronto puzzle, a version of the Tower of Hanoi puzzle. Seventy-two healthy volunteers took part in this double-blind study. Chlorpromazine spared free-recall and word-completion performance, but impaired the acquisition of a cognitive routine in the subjects who completed the first trials of the Tower of Toronto puzzle efficiently. Lorazepam induced an opposite pattern of memory disruption. These preliminary results suggest that chlorpromazine and lorazepam induced a double dissociation between priming and the acquisition of a cognitive routine. They provide evidence that the two forms of implicit memory rely upon distinct neurochemical systems, the latter, but not the former, being dependent upon dopaminergic systems.

[Paroxysmal neurological manifestations disclosing panic attacks]. / Manifestations neurologiques paroxystiques révélatrices d'attaques de panique.

Thirty-seven patients presented with paroxysmal neurological manifestations attributed to anxiety attacks. The manifestations included loss of consciousness, focal sensorimotor deficits, diffuse dysesthaesiae, visual disorders and tremor. They lasted 10 to 45 minutes and occurred once per day to once per week. Organic pathology was dismissed on the basis of normal examinations and atypical course. In all patients questioning revealed symptoms that were those of acute anxiety. The fact that these attacks took place in suggestive (circumstances e.g. in crowds and car driving), and that they could be induced by challenge tests hyperpnoea, infusion of lactate) suggested that these disorders were consecutive to panic attacks.

Construction of a specialized-ribosome vector or cloned-gene expression in E. coli.

An expression system utilizing specialized ribosomes has been constructed with beta-galactosidase as the product. Ribosomes specific for lacZ mRNA are generated due to a mutation within the anti-Shine-Dalgarno region of a plasmidborne 16S rRNA gene that is complementary to a mutation within the ribosome-binding site of lacZ. Hence, a subpopulation of ribsomes specific for translation of the cloned gene mRNA is produced. Transcription of the lacZ gene is regulated by the tac promoter, while transcription of the mutated rrnB locus is controlled by the lambdaP(L) promoter. Batch experiments indicate that full induction of both operons (2 mM IPTG, 42 degrees C) leads to maximal beta-galactosidase activity per cell at levels 35% higher than that obtained using a wild-type ribosome expression system. Using a novel, site-directed mutagenesis technique, construction of the specialized ribosome vector is outlined, and the results of lacZ expression are presented as transcription of both the cloned-gene and the specialized-ribosome locus are induced.

Microfluorimetric analysis of spatial and temporal patterns of immobilized cell growth.

Pronounced spatial nonuniformities in cell density, physiology, and activity frequently arise within densely packed immobilized cell supports. For a more fundamental understanding of immobilized cell phenomena, we have developed high-resolution microfluorimetric procedures to analyze local variations in both immobilized cell loading and growth rate. Fluorescent staining of total cellular DNA provides a measure of local biomass density. Actively growing (DNA synthesizing) cells are marked by pulse-labeling newly synthesized DNA with the thymine analog, bromouracil. An immunofluorescent technique allows subsequent detection of spatial variations in DNA synthesis rates. These procedures enable the influence of mass-transfer limitations and other immobilization effects on cell distribution and activity to be readily quantified. We demonstrate this approach through analysis of the patterns of growth of Escherichia coli entrapped within Sr-alginate gel beads. The experimental techniques are potentially applicable to a variety of other aggregate cell systems.

Effect of chemically-induced, cloned-gene expression on protein synthesis in E. Coli.

Earlier experiments in our lab investigated the metabolic limitations of cloned-gene expression in bacterial cells (for over-production of beta-lactamase). These experiments showed that the steady-state concentration of ribosomal RNA decreased upon plasmid amplification while both the synthesis rate and steady-state beta-lactamase mRNA level increased significantly. This appeared to indicate substantial limitation exist within the transnational machinery of the bacterial cell at high copy numbers. To establish the generality of this phenomenon, the impact increasing protein expression from pa plasmid by chemically inducing a strong promoter while maintaining constant copy number has been investigated. A plasmid has been constructed which contains the lacZ gene under control of the tac promoter and contains the parB stability locus to maintain plasmid stability. Using this vector, beta-galactosidase expression in chemostat cultures operated at specific growth rates of 0.6 h(-1) was induced with IPTG such that enzyme activity was varied over a 460-fold range. When fully induced beta-galactosidase protein production represented 14 wt % of total cell protein. As transcription was induced, the synthesis rate of the beta-galactosidase mRNA increased 42-fold while the steady-state level of beta-galactosidase mRNA increased only fourfold. This indicates stability may play a larger role for beta-galactosidase expression with a strong promoter than seen with beta-lactamase production in the elevated copy number system. Furthermore, rRNA synthesis rates increased at high expression rates as seen in the copy number experiments. However, unlike the amplified-plasmid system, the steady-state levels of rRNA increased as well. Since the total protein levels closely followed the steady-state level of eRNA, transnational limitations are again suggested for the chemically induced transcription system.