Rapid screening for metabolite overproduction in fermentor broths, using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks

Binary mixtures of model systems consisting of the antibiotic ampicillin with either Escherichia coli or Staphylococcus auresu were subjected to pyrolysis mass spectrometry (PyMS). To deconvolute the pyrolysis mass spectra, so as to obtain quantitative information on the concentration of ampicilin in the mixtures, partial least squares regression (PLS), principal components regression (PCR), and fully interconnected feedforward artificial neural networks (ANNs) were studied. In the latter case, the weights were modified using the standard backpropagation algorithm, and the nodes used a sigmoidal squsahing funciton. It was found that each of the methods could be used to provide calibration models which gave excellent predictions for the concentrations of ampicillin in samples on which they had not been trained. Furthermore, ANNs trained to predict the amount of ampicilin in E. coli were able to generalise so as to predict the concentration of ampicillin in a S. aureus background, illustrating the robustness of ANNs to rather substantial variations in the biological background. The PyMS of the complex mixture of ampicilin in bacteria could not be expressed simply in terms of additive combinations of the spectra describing the pure components of the mixtures and their relative concentrations. Intermolecular reactions took place in the pyrolysate, leading to a lack of superposition of the spectral components and to a dependence of the normalized mass spectrum on sample size. Samples from fermentations of a single organism in a complex production medium were also analyzed quantitatively for a drug of commercial interest. The drug could also be quantified in a variety of mutant-producing strains cultivated in the same medium. The combination of PyMS and ANNs constitutes a novel, rapid, and convenient method for exploitation in strain improvement screening programs. (c) 1994 John Wiley & Sons, Inc.     

Craniometrical variations among eastern Brazilian marmosets and their systematic relationships

The craniometrical variations among marmosets on the eastern coast of Brazil or theCallithrix jacchus group (C. jacchus, C. penicillata, C. kuhli, C. geoffroyi, C. flaviceps, andC. aurita) were analyzed to test various hypotheses for the arrangement of the species and phylogenetic relationships ofC. kuhli andC. flaviceps. Q-mode correlation coefficients and principal component analysis were employed to extract important shape factors from the craniometrical data. On this basis, the shape distances among the eastern Brazilian marmosets were found to be larger than those between the marmosets which are recognized as distinct species. This evidence supports the hypothesis that the members of theC. jacchus group are distinct species.C. kuhli shows the strongest resemblance toC. geoffroyi within theC. jacchus group.C. flaviceps is the most similar toC. aurita in its cranial shape. These findings indicate thatC. kuhli is most closely related toC. geoffroyi, whileC. flaviceps is toC. aurita.     

厌氧消化过程的非模型控制

对废水淤泥的厌氧消化处理过程进行了研究．提出了一种基于神经网络的非模型控制方法。多变量控制系统的操作变量为供热量和进水量．被控变量为消化温度和消化污泥排出浓度。证明了控制算法的收敛性。讨论了控制系统的稳定性。仿真结果证实了非模型控制方法的有效性。该方法无需对象的模型．为复杂生化过程的控制提供了一种新途径。     

Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts

The dopamine (DA) uptake system was investigated in the mesostriatal system of normal and weaver mutant mice, which lose mesencephalic DA neurons, as well as in weaver mutants with ventral mesencephalic grafts to the striatum. Assays of [³H]DA uptake in striatal synaptosomal fractions in vitro and autoradiography of [³H]mazindol binding in brain sections were carried out in wild-type mice (+/+) and in the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Net [³H]DA uptake, expressed as pmol/mg-protein/2-min, was on the average 50.6 in the striatum of wild-type mice, 7.9 in the non-grafted, and 10.1 in the transplanted striatum of weaver mutants. [³]DA uptake in wild-type mice differed significantly from both the grafted and non-grafted weaver striata (P<0.001). Paired comparisons for [³H]DA uptake between right and left sides of recipient weaver mice showed a significant side effect (P<0.02), the right side being 28–38% higher than the left side [mean of all individual (R-L)/L values]. The results of amphetamine-induced turning behavior tests were compared with the biochemical findings. Mice with grafts to the right side rotated an average of 22 turns to the left and 7 turns to the right during the five one-minute sessions; the mean value L/(L+R) was 64%. A plot of (L-R) rotations against (R-L) [³H]DA uptake gave a correlation coefficient of 0.552 (P<0.05), indicating that animals with a strong rotational bias to the left tended to have higher [³H]DA on the right. Similarly, the animals that were used for [³H]mazindol binding autoradiographic studies displayed on the average 72% rotations to the left side. In the [³H]mazindol binding data, non-grafted weaver mutants showed the severest depletion relative to wild-type in the dorsomedial and dorsolateral caudate-putamen (86% and 87%, respectively). Mice with unilateral grafts to the right side showed an increase in [³H]mazindol binding signal in the transplanted side of 40–64% (depending on dorsoventral topography) over the contralateral, non-grafted side. These findings attest to the functional effects of the grafts at the anatomical, biochemical, and behavioral levels. The parallel measurements of motor performance and DA uptake in the same animals offers an index of behavioral recovery as a function of transmitter-related activity. Furthermore, by conducting measurements of the synaptosomal DA uptake in vitro and of the binding characteristics of mazindol in brain slices by autoradiography, one has the advantage of combining the anatomical resolution of uptake site visualization with a dynamic indicator of function for DA uptake in the nerve terminal.Special issue dedicated to Professor Sidney Ochs     

Target determination of neurotransmitter phenotype in sympathetic neurons

While the majority of sympathetic neurons are noradrenergic, a minority population are cholinergic. At least one population of cholinergic sympathetic neurons arises during development by a target-dependent conversion from an initial noradrenergic phenotype. Evidence for retrograde specification has been obtained from transplantation studies in which sympathetic neurons that normally express a noradrenergic phenotype throughout life were induced to innervate sweat glands, a target normally innervated by cholinergic sympathetic neurons. This was accomplished by transplanting footpad skin containing sweat gland primordia from early postnatal donor rats to the hairy skin region of host rats. The sympathetic neurons innervating the novel target decreased their expression of noradrenergif traints and developed choline acetyltransferase (ChAT) activity. In addition, many sweat gland-associated fibers acquired acetylcholinesterase (AChE) staining and VIP immunoreactivity. These studies indicated that sympathetic neurons in vivo alter their neurotransmitter phenotype in response to novel envronmental signals and that sweat glands play a critical role in the cholinergic and peptidergic differentiation of the sympathetic neurons that innervate them. The sweat gland-derived cholinergic differentiation factor is distinct from leukemia inhibitory factor and ciliary neurotrophic factor, two well-characterized cytokines that alter the neurotransmitter properties of cultured sympathetic neurons in a similar fashion. Recent studies indicate that anterograde signalling is also important for the establishment of functional synapses in this system. We have found that the production of cholinergic differentiation activity by sweat glands required sympathetic innervation, and the acquisition and maintenance of secretory competence by sweat glands depends upon functional cholinergic innervation. 1994 John Wiley & Sons, Inc.     

Occurrence,distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.     

Expanding EMC foldopathies: Topogenesis deficits alter the neural crest

The endoplasmic reticulum (ER) membrane protein complex (EMC) is essential for the insertion of a wide variety of transmembrane proteins into the plasma membrane across cell types. Each EMC is composed of Emc1-7, Emc10, and either Emc8 or Emc9. Recent human genetics studies have implicated variants in EMC genes as the basis for a group of human congenital diseases. The patient phenotypes are varied but appear to affect a subset of tissues more prominently than others. Namely, craniofacial development seems to be commonly affected. We previously developed an array of assays in Xenopus tropicalis to assess the effects of emc1 depletion on the neural crest, craniofacial cartilage, and neuromuscular function. We sought to extend this approach to additional EMC components identified in patients with congenital malformations. Through this approach, we determine that EMC9 and EMC10 are important for neural crest development and the development of craniofacial structures. The phenotypes observed in patients and our Xenopus model phenotypes similar to EMC1 loss of function likely due to a similar mechanism of dysfunction in transmembrane protein topogenesis.