A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Substance P and enkephalin in transected axons of medulla and spinal cord

The accumulation of transported materials in cut axons is demonstrated by the light and electron microscopic immunocytochemical localization of substance P and enkephalin in the caudal medulla and cervical spinal cord of adult rat. Two days following unilateral knife-cuts in the caudal medulla or spinal (C2-C3) levels, substance P and enkephalin-like immunoreactivity (SPLI and ELI) are detected in lesioned axons located rostral and caudal to the transection. Rostrally, SPLI and ELI are detected in the lateral reticular region and ventrolateral fasciculus corresponding to the location of previously identified bulbospinal pathways. Caudally, previously unidentified, propriospinal pathways showing SPLI are detected in the dorsal columns and in the dorsolateral fasciculus. In contrast, ELI is found caudal to the transection only in the reticular region of the medulla. For both peptides, immunoreactivity is present throughout axons containing numerous large, dense core, and small clear vesicles. These results support the concept of both particulate and soluble modes of transport for substance P and enkephalin within axons of the central nervous system.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Regulation of UDP-Galactose:Ceramide Galactosyltransferase and UDP-Glucose:Ceramide Glucosyltransferase After Crush and Transection Nerve Injury

The enzyme activities of ceramide galactosyltransferase and ceramide glucosyltransferase were assayed as a function of time (0, 1, 2, 4, 7, 14, 21, 28, and 35 days) after crush injury or permanent transection of the adult rat sciatic nerve. These experimental models of neuropathy are characterized by the presence and absence of axonal regeneration and subsequent myelin assembly. Within the first 4 days after both injuries, a 50% reduction of ceramide galactosyltransferase-specific activity was observed compared to values found in the normal adult nerve. This activity remained unchanged at 7 days after injury; however, by 14 days the ceramide galactosyltransferase activity diverged in the two models. The activity increased in the crushed nerve and reached control values by 21 days, whereas a further decrease was observed in the transected nerve such that the activity was nearly immeasurable by 35 days. In contrast, the ceramide glucosyltransferase activity showed a rapid increase between 1 and 4 days, followed by a plateau that was 3.4-fold greater than that in the normal adult nerve, which persisted throughout the observation period in both the crush and transection models. [3H]Galactose precursor incorporation studies at 7, 14, 21, and 35 days after injury confirmed the previously observed shift in biosynthesis from the galactocerebrosides during myelin assembly in the crush model to the glucocerebrosides and oligohexosylceramide homologues in the absence of myelin assembly in the transection model. The transected nerves were characterized by a peak of biosynthesis of the glucocerebrosides at 14 days. Of particular interest is the biosynthesis of the glucocerebrosides and the oligohexosylceramides at 7 and 14 days after crush injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

三叉神经中脑核神经元膜的电学特性

用大鼠脑干脑片,给三叉神经中脑核79个神经元作了细胞内记录,测算了20个神经元膜的电学特性:静息电位-60.3±5.6mV;输入阻抗为10.5±5.4MΩ;时间常数1.3±0.5ms。电刺激可诱发动作电位,测算32个神经元的有关参数:阈电位-50—-55mV;波幅69.5±6.1mV;超射11.9±3.6mV;波宽0.8±0.2ms。TTX(0.3μmol/L)或无钠使之消失。通以长时程矩形波电流可引起200—250Hz的2—15个重复放电,但在通电停止前终止,TEA或4-AP可延长放电。膜电位-60—-55mV时在动作电位之后可看到阈下电位波动,它不受TTX的影响,无钙时消失,TEA或4-AP使波幅增大。静息电位去极化可使45个神经元中的40个发生外向整流作用,并被TEA,4-AP或无钙抑制,超极化则发生内向整流作用,Cs或无钠抑制之。灌流液中加入各种钾通道阻断药时神经元的稳态I-V曲线发生相应变化,提示I_(DR),l_A,I_(K(Ca))及I_Q可能都与静息时的膜电导有关。     

Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of Injury-Induced Calcium Entry into Peripheral Nerve Myelinated Axons: Role of Reverse Sodium-Calcium Exchange

Abstract: To investigate the route of axonal Ca²⁺ entry during anoxia, electron probe x-ray microanalysis was used to measure elemental composition of anoxic tibial nerve myelinated axons after in vitro experimental procedures that modify transaxolemmal Na⁺ and Ca²⁺ movements. Perfusion of nerve segments with zero-Na⁺/Li⁺-substituted medium and Na⁺ channel blockade by tetrodotoxin (1 µM) prevented anoxia-induced increases in Na and Ca concentrations of axoplasm and mitochondria. Incubation with a zero-Ca²⁺/EGTA perfusate impeded axonal and mitochondrial Ca accumulation during anoxia but did not affect characteristic Na and K responses. Inhibition of Na⁺-Ca²⁺ exchange with bepridil (50 µM) reduced significantly the Ca content of anoxic axons although mitochondrial Ca remained at anoxic levels. Nifedipine (10 µM), an L-type Ca²⁺ channel blocker, did not alter anoxia-induced changes in axonal Na, Ca, and K. Exposure of normoxic control nerves to tetrodotoxin, bepridil, or nifedipine did not affect axonal elemental composition, whereas both zero-Ca²⁺ and zero-Na⁺ solutions altered normal elemental content characteristically and significantly. The findings of this study suggest that during anoxia, Na⁺ enters axons via voltage-gated Na⁺ channels and that subsequent increases in axoplasmic Na⁺ are coupled functionally to extraaxonal Ca²⁺ import. Intracellular Na⁺-dependent, extraaxonal Ca²⁺ entry is consistent with reverse operation of the axolemmal Na⁺-Ca²⁺ exchanger, and we suggest that this mode of Ca²⁺ influx plays a general role in peripheral nerve axon injury.     

Neurotrophin-4: The odd one out in the neurotrophin family

Neurotrophin-4 (NT-4) is a member of a family of neurotrophic factors, the neurotrophins, that control survival and differentiation of vertebrate neurons (2–4). Besides being the most recently discovered neurotrophin in mammals, and the least well understood, several aspects distinguish NT-4 from other members of the neurotrophin family. It is the most divergent member and, in contrast to the other neurotrophins, its expression is ubiquitous and appears to be less influenced by environmental signals. NT-4 seems to have the unique requirement of binding to the lowaffinity neurotrophin receptor (p75^LNGFR) for efficient signalling and retrograde transport in neurons. Moreover, while all other neurotrophin knock-outs have proven lethal during early postnatal development, mice deficient in NT-4 have so far only shown minor cellular deficits and develop normally to adulthood. Is NT-4 a recent addition to the neurotrophic factor repertoire in search of a crucial function, or is it an evolutionary relic, a kind of wisdom tooth of the neurotrophin family?