几种抗菌药物对腹泻羔羊肠道细菌的影响及临床观察

通过对治疗前后腹泻羔羊粪便细菌检查及临床观察,以查明两种抗菌药物对腹泻羔羊肠道细菌的影响。结果表明,腹泻羔羊治疗前粪便中的革兰氏阳性杆菌,革兰氏阳性球菌及革兰氏阴性杆菌的比例分别为20％、10％和70％。用敌菌净治疗的效果显著,其羔羊肠道细菌的比例接近健康羔羊。用庆大霉素治疗的羔羊,部分出现不良反应,其肠道细菌出现失调状态。     

Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of newborn rats

Summary The changes in Na current during development were studied in the dorsal root ganglion (DRG) cells using the whole-cell patch-clamp technique. Cells obtained from rats 1–3 and 5–8 days after birth were cultured and their Na currents were compared. On top of the two types of Na currents reported in these cells (fast-FA current and slow-S current) a new fast current was found (FN). The main characteristics of the three currents are: (i) The voltages of activation are –37, –36, and –23 mV for the FN, FA and S currents, respectively. (ii) The activation and inactivation kinetics of FN and FA currents are about five times faster than those of the S current. (iii) The voltages at which inactivation reaches 50% are –139, –75 and –23 mV for the FN, FA and S currents, respectively.The kinetics and voltage-dependent parameters of the three currents and their density do not change during the first eight days after birth. However, their relative frequency in the cells changes. In the 1–3 day-old rats the precent of cells with S, FA, and mixed S+FN currents is 22, 18, and 60% of the cells, respectively. In the 5–8 day-old, the percent of cells with S, FA, and FN+S is 10, 66 and 22%. The relative increase in the frequency of cells with FA current during development can contribute to the ease of action potential generation compared with cells with FN currents, which are almost completely inactivated under physiological conditions. The predominance of FA cells also results in a significant decrease in the relative frequency of cells with the high-threshold, slow current.Antibodies directed against a part of the S₄ region of internal repeat I of the sodium channel (C ₁ ⁺ , amino acids 210–223, eel channel numbering) were found to shift the voltage dependence of FA current inactivation (but not of FN or S currents) to more negative potentials. The effect was found only when the antibodies were applied externally. The results suggest that FN, FA and S types of Na currents are generated by channels, which are different in the topography of the C ₁ ⁺ region in the membrane.     

Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.     

Overexpressions of cDNAs for β-Amyloid Precursor Proteins 695, 751, and 770 Enhance the Secretion of β-Amyloid Precursor Protein Derivatives and the Survival of P19-Derived Neurons

Abstract: P19 is a C3H mouse-derived line of multipotent embryonic carcinoma cells that differentiate into neural cells. P19 cell clones overexpressing the three major forms of β-amyloid precursor protein from their cDNA constructs were established. Unlike a previous study in which P19-derived neurons had a limited α-secretase activity, all of these clones produced significant amounts of secreted β-amyloid precursor protein. When treated with retinoic acid, these transformed lines differentiated into neurons and survived better than did nontransformed parental P19 cells. Furthermore, P19-derived neurons survived better in medium conditioned by the transformed P19 line, and survival was reduced by immunoabsorption with an antibody to β-amyloid precursor protein. These results suggest neurotrophic effects of secreted β-amyloid precursor protein and contrast with a previous report in which overexpression of a full-length cDNA for β-amyloid precursor protein led to degeneration of P19-derived neurons. Western blot analysis suggested that this difference might result from different levels of expression of putative neurotoxic C-terminal fragments of β-amyloid precursor protein; moreover, P19-derived neurons differ from P19 stem cells in the processing of these C-terminal fragments.     

Neuropeptide Y perfused in the preoptic area of rats shifts extracellular efflux of dopamine,norepinephrine and serotonin during hypothermia and feeding

This study examined the localized action of neuropeptide Y (NPY) on monoamine transmitter activity in the hypothalamus of the unrestrained rat as this peptide induced hypothermia, spontaneous feeding or both responses simultaneously. A guide tube was implanted in the anterior hypothalamic pre-optic area (AH/POA) of Sprague-Dawley rats. Then either control CSF vehicle or NPY in a dose of either 100 ng/μl or 250 ng/μl was perfused by push-pull cannulae in this structure in the fully sated, normothermic rat. Successive perfusions were carried out at a rate of 20 μl/min for 6.0 min with an interval of 6.0 min elapsing between each. Samples of perfusate were assayed by HPLC for their levels of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their respective metabolites. Whereas control CSF was without effect on body temperature (T_b) or feeding, repeated perfusions of NPY over 3.0 hr caused dose—dependent eating from 4 to 39 g of food, hypothermia of 0.9 to 2.3°C or both responses concurrently. As the rats consumed 11–39 g of food, the efflux of NE, MHPG, DOPAC and 5-HT was enhanced significantly, whereas during the fall in T_b the efflux of NE, DOPAC and 5-HIAA from the AH/POA increased. When the T_b of the rat declined simultaneously with eating behavior, the levels in perfusate of DOPAC and HVA increased significantly while MHPG declined. During perfusion of the AH/POA with NPY the turnover of NE declined while DA and 5-HT turnover increased during hypothermia alone or when accompanied by feeding. These results demonstrate that the sustained elevation in NPY within the AH/POA causes a selective alteration in the activity of the neurotransmitters implicated in thermoregulation, satiety and hunger. These findings suggest that both DA and NE comprise intermediary factors facilitating the action of NPY on neurons involved in thermoregulatory and ingestive processes. The local activity of NPY on hypothalamic neurons apparently shifts the functional balance of serotonergic and catecholaminergic neurons now thought to play a primary role in the control of energy metabolism and caloric intake.     

Prediction of location of active sites in biologically active peptides

In a previous paper we demonstrated that the short-range compact regions in atrial natriuretic factor (-hANF) predicted by the average distance map (ADM) correspond to its active sites [Kikuchi,J. Protein Chem.11, 579–581 (1992)]. In the present paper we apply the same method to other bioactive peptides and peptidic enzyme inhibitors. We again observe that active sites in each peptide are contained in short-range compact regions predicted by the ADM for the peptide. This demonstrates that the ADM method predicts the possible location of active sites in biologically active peptides in general. The possibility of practical application of the present method to rational drug design is also discussed.     

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

Ontogeny of some endocrine cells of the digestive tract in sea bass (Dicentrarchus labrax): An immunocytochemical study

Serotonin- and ten peptide-immunoreactive (IR) cell types were identified in the digestive tract of sea bass (Dicentrarchus labrax L.) larvae of four morphofunctional phases ranging in age from hatching to 61 days. The sequence of appearance and location of endocrine cells during ontogenetic development of the larvae was determined. The differentiation of endocrine cells followed a distal-proximal gradient in the gut which paralleled the morphofunctional differentiation. Serotonin-IR cells were identified in the last portion of the digestive tract from phase I onwards and in the gastric region from phase III, before these regions were morphofunctionally differentiated; met-enkephalin-IR cells were identified from phase II onwards in both the differentiated rectum and the undifferentiated intestine; cholecystokinin (CCK)- and synthetic human gastrin-34-IR cells were located only in the intestine and first found in the undifferentiated intestine of phase II; human gastrin-17-, peptide YY (PYY)- and neuropeptide Y (NPY)-IR cells appeared in the intestine from phase II and in stomach in phase IV, when it showed gastric glands; pancreatic polypeptide (PP)- and glucagon-IR cells were observed in both intestine and stomach, but insulin- and somatostatin-IR cells only in stomach, from phase III, during which the intestine but not the stomach was differentiated. PP- and PYY-, PP- and glucagon-, and PYY- and glucagon-like immunoreactivities coexisted from their first appearance in some cells of the gut.     

Effects of amphipathic peptides,including presequences,on the functional integrity of rat liver mitochondrial membranes

A number of amphipathic peptides were tested for their effects on structural and functional properties of isolated rat liver mitochondria. The peptides included the matrix targeting sequence of subunit IV of (yeast) cytochromec oxidase. Titration experiments in which the mitochondria were incubated with increasing concentrations of the peptides revealed two major stages in the interaction. First, at low peptide/mitochondria ratios, peptide binding to the outer membrane occurred which was accompanied by gradual lysis of the outer membrane at higher ratios. The latter was deduced from the release of adenylate kinase, the classical marker enzyme of the intermembrane space. Secondly, at still higher peptide/mitochondria ratios, the permeability of the inner membrane progressively increased, as evidenced by measurements of respiratory control and of the membrane potential. Complete uncoupling of respiration seemed to precede dissipation of the membrane potential.     

A facile method to prepare C-terminal fluorescently labelled peptides by an Fmoc strategy

Summary Spectrophotometric peptide probes, derivatized at the C-terminus, are conveniently prepared by means of an Fmoc solid-phase strategy. Using a resin such as Sasrin, the fully protected peptide can be cleaved from the resin with hydrazine, yielding the protected peptide-hydrazide which is subsequently oxidized to the azide. An amino-containing chromophore or fluorophore such as 5-[(2-aminoethyl)-amino]-naphthalene sulfonic acid (EDANS) can be coupled directly to this activated carboxyl group. This allows for specific placement of the fluorophore at the C-terminal carboxyl group in the presence of trifunctional amino acids.