Proteins of fast axonal transport in the regenerating hypoglossal nerve of the rat

The composition of proteins conveyed by fast axonal transport in growing or regenerating axons is different from that of intact, mature axons. Consistent alterations have been observed in several different types of neurons, but adult peripheral axons (rabbit hypoglossal motoneurons) seemed to be exceptions because during their regeneration there was no increased labelling of a 23 kilodalton (kD) protein associated with the growth state. We examined the composition of fast-transported proteins, labelled by application of [35S]methionine to the hypoglossal nuclei, in intact and regenerating hypoglossal nerves of the rat. Using one- and two-dimensional electrophoresis we detected both increases and decreases in the labelling of specific polypeptides during regeneration. In particular, there was increased labelling of a 23 kD polypeptide. Changes were maximal 7 days after axotomy and subsided thereafter, coincident with reinnervation of the tongue. We conclude that hypoglossal axons show the same changes in transported protein composition which are characteristic of the growth state in other axons. Thus, we have strengthened the correlation between the growth state and changes in synthesis of a set of polypeptides of unknown function.     

Effect of partial ischemia and axotomy on activities of cholinergic enzymes in lower motor neurons of the dog

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the ventral spinal cord, ventral spinal roots and in the central and peripheral stumps of the sciatic nerve transected under conditions of partial ischemia (produced by aortic ligation just below the renal arteries) were compared to those obtained under intact blood supply in time intervals 5, 10, or 15 days after surgery. The significant increase of ChAT activity in the central part of the sciatic nerve following 15 days of partial ischemia correlated with less significant elevation of ChAT in the ventral spinal cord. The changes of AChE activity were not significant during partial ischemia. ChAT in the peripheral stump of the sciatic nerve following 5 days of partial ischemia was preserved by 40% and AChE by 20% more than under normal blood supply. On the contrary, in the next 5 days interval losses of enzymes activity in the degenerating nerve were greater. ChAT was almost totally inactivated whereas 50% of AChE activity was preserved until the end of period examined.     

Raman optical activity of proteins, carbohydrates and glycoproteins

On account of its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the structure of biomolecules. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration and side-chain conformations, and on structural elements present in unfolded states. Carbohydrate ROA spectra provide information on the central features of carbohydrate stereochemistry, especially that of the glycosidic link. Glycoprotein ROA spectra provide information on both the polypeptide and carbohydrate components. This article describes the ROA technique and presents and discusses the ROA spectra of a selection of proteins, carbohydrates, and a glycoprotein. The many structure-sensitive bands in protein ROA spectra are favorable for applying pattern recognition techniques, illustrated here using nonlinear mapping, to determine structural relationships between different proteins.     

Unstable nuclear DNA in hypoglossal neurons of adult mice

To demonstrate the existence of unstable or metabolic DNA in normal mammalian neurons and to study the effect of peripheral nerve injury on this metabolic DNA, adult mice were given repeated injections of high doses of 3H-thymidine (3H-T) on the day before injury to the left hypoglossal nerve. The animals were killed at intervals up to 33 days after the injections of 3H-T. Analyses of grain counts showed a low but significant elevation in the number of radioautographic grains per unit area of hypoglossal neuronal nuclei above background levels for up to 5 days after 3H-T injection. Digestion of the tissue with DNase lowered the nuclear grain counts to background levels, confirming that the DNA was indeed labelled. Although there was a loss of labelled material from the neuronal nuclei with time, there was no difference between injured and uninjured neurons at any of the intervals tested after injection of 3H-T.     

Inhibitors of the sulfation of proteins, glycoproteins, and proteoglycans

Two categories of compounds, substrates of sulfation and sulfate analogs, were tested for the ability to inhibit sulfation of macromolecules secreted by HepG2 cells. Several compounds which most effectively inhibited sulfation without toxic effects on cells were tested for their relative inhibition of sulfation of tyrosine residues (using the fourth component of complement as a model substrate), of N-linked oligosaccharides (alpha 2HS-glycoprotein as substrate), and of proteoglycans. Inhibitors decreased the sulfation of all three classes of substrate, but not always equally. Use of inhibitors from both categories in combination yielded synergistic effects, with more effective inhibition of sulfation and low toxicity. Such combinations of inhibitors should provide a valuable tool for probing the significance of the sulfation of macromolecules.     

Rates of protein synthesis in the regenerating hypoglossal nucleus: Effects of testosterone treatment

Rates of protein synthesis (lCPS_leu) along the entire rostral to caudal extent of the hypoglossal nucleus were determined in adult, female rats with the quantitative autoradiographicl-[1-¹⁴C]leucine method two and five weeks after unilateral hypoglossal axotomy with and without chronic treatment with testosterone. Rates of protein synthesis were increased on the axotomized side, and the increases were greater in the rostral portion of the nucleus at both time points examined. The effects of axotomy on lCPS_leu were less at five weeks post-axotomy than at two weeks. In spite of the fact that testosterone has been shown to accelerate both the rate of outgrowth of regenerating cranial motor nerves (Kujawa et al., J. Neurosci. 113898–3906, 1991) and the recovery of function (Kujawa et al., Exp. Neurol. 10580–85, 1989) and to attenuate the loss of neurons (Yu et al., Exp. Neurol. 80349–360, 1983) there were no effects of testosterone on 1CPS_leu in the hypoglossal nucleus in either sham-operated or axotomized rats.Abbreviations Used ie The local cerebral rate of protein synthesis - {ie623-1} the rate of leucine incorporation into protein is abbreviated as lCPS_leu - i is the fraction of leucine in the precursor pool for protein synthesis derived from the arterial plasma in tissue region - TP is testosterone propionate Special issue dedicated to Dr. Bernard W. Agranoff.     

The salubrious effect of tamaxifen on serum marker enzymes,glycoproteins, and lysosomal enzymes level in breast cancer women

Tumour markers correlate strongly with prognosis based on tumour burden and surgical resectability. If chemotherapy is extremely effective in certain stage of the disease, the sensitive marker may be of great use in monitoring disease response and drug treatment. Hence, this study was launched to evaluate the changes in tumour marker enzymes like lactate dehydrogenase (LDH), glumate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase, and acid phosphatase in before and after 3 and 6 months tamoxifen treated breast cancer patients. In addition, the changes in serum glycoproteins viz., hexose, hexosamine, and sialic acid and lysosomal enzymes such as N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were analysed in these patients. These values were compared with their age matched healthy control subjects. At 6 months evaluation, the tamoxifen treated postmenopausal breast cancer women showed a statistically significant decreased (p < 0.001, 0.05 respectively) levels of LDH, SGOT, SGPT, alkaline and acid phosphatases than their baseline values. Similarly, the levels of hexose, hexosamine, and sialic acid and N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were decreased significantly (p < 0.001 ) in tamoxifen received postmenopausal women. The result of this study suggested that tamoxifen potentially retard the metastasis of breast cancer as well as the bone demineralisation in postmenopausal breast cancer women. Thus, tamoxifen may also have its antitumour activity through its beneficial effects on tumour marker enzymes and serum proteins in breast cancer women.     

A difference between the proteins conveyed in the fast component of axonal transport in guinea pig hypoglossal and vagus motor neurons

We compared the proteins transported in the fast component of guinea pig hypoglossal motor neurons with those of guinea pig vagus (preganglionic parasympathetic) neurons. The fast component proteins of hypoglossal and vagus neurons were radioactively labeled by injecting ³H-amino acids into the hypoglossal and vagus motor nuclei. The radioactive fast component proteins obtained from each system were then compared with each other by SDS-polyacrylamide slab gel electrophoresis and fluorography. These analyses revealed at least twenty polypeptides which appear common to the fast component of each neuronal system. In addition, we identified one difference between the proteins comprising the fast component of these neuronal systems. A polypeptide, molecular weight 50,000 daltons, present in the fast component of vagus neurons was not detected in the fast component of hypoglossal motor neurons. These observations are discussed with regard to the similarities and differences between these neuronal systems.     

Kainate receptors and RNA editing in cholinergic neurons

Parasympathetic ganglia are considered simple relay systems that have cholinergic input and output, with modulation occurring centrally. Greater complexity is suggested, however, by our showing here that avian ciliary ganglion (CG) neurons also express a different excitatory receptor type--ionotropic glutamate receptors of the kainate subtype (KARs). This is the first report of glutamate receptor expression in the CG and KAR expression in any cholinergic neuron. We show that KARs form functional channels on CG neurons. KARs localize to CG neuron axons and somata as well as axons and terminals of pre-synaptic inputs to the CG. Glutamate transporters are expressed on Schwann cells that surround synapses on neuronal somata, and may provide a local source of glutamate. CG neurons express multiple KAR subunit mRNAs (GluR5, GluR7, and KA1), and their relative levels change dramatically during axon outgrowth and synaptic differentiation. The developmental role for KARs may depend upon their calcium permeability, a property regulated by mRNA editing. We show GluR5 editing increases predominantly at the time CG axons contact peripheral targets. Our data suggest that glutamatergic signaling may function as a local circuit mechanism to modulate excitability and calcium signaling during synapse formation and maturation in the CG in vivo.     

A difference between the proteins conveyed in the fast component of axonal transport in guinea pig hypoglossal and vagus motor neurons.

We compared the proteins transported in the fast component of guinea pig hypoglossal motor neurons with those of guinea pig vagus (preganglionic parasympathetic) neurons. The fast component proteins of hypoglossal and vagus neurons were radioactively labeled by injecting 3H-amino acids into the hypoglossal and vagus motor nuclei. The radioactive fast component proteins obtained from each system were then compared with each other by SDS-polyacrylamide slab gel electrophoresis and fluorography. These analyses revealed at least twenty polypeptides which appear common to the fast component of each neuronal system. In addition, we identified one difference between the proteins comprising the fast component of these neuronal systems. A polypeptide, molecular weight 50,000 daltons, present in the fast component of vagus neurons was not detected in the fast component of hypoglossal motor neurons. These observations are discussed with regard to the similarities and differences between these neuronal systems.     

Differential display cloning of genes induced in regenerating neurons

Mammalian adult motor and sensory neurons are thought to reexpress a complement of genes that are originally expressed during development when they regenerate following injury. Therefore, one strategy for identifying key genes involved in development of the peripheral nervous system is to identify those genes reexpressed in the regenerating system. To test this hypothesis, we used the single-base anchor method of mRNA differential display to study changes in gene expression in regenerating adult mammalian sensory neurons. From an initial sample of 36 different primer combinations [3 oligo(dT)M primers x 12 arbitrary 13-mers], 6 candidate upregulated and 6 candidate downregulated genes were identified. Candidate genes were screened by the reverse Northern blot method to eliminate obvious false positives and the three remaining candidates cloned and sequenced. In addition to comparing isolated sequences with the public databases, sequences were also compared with assembled clusters of expressed sequence tag sequences, enabling extension of the sequence data by more than a kilobase from the isolated 3' cDNA fragments. Ultimate confirmation of differential expression was carried out by in situ hybridization using 45-base oligonucleotides complementary to the predicted 5'-3' orientation of the corresponding mRNAs of all three cDNAs. Two, LA12.2 and LC12, were definitively confirmed as induced in regenerating neurons. The sequence of LC12 is identical to that of the secreted protein Reg-2 and a detailed study of the functions of this secreted protein in neural development and regeneration has been published (F. J. Livesey, J. A. O'Brien, M. Li, A. G. Smith, L. J. Murphy, and S. P. Hunt, 1997, Nature 390, 614-618). The LA12.2 gene is currently being characterized, the available sequence of this cDNA being novel.     

Cell death of asynaptic neurons in regenerating spinal cord

The weakly electric fish Sternarchus albifrons possesses a unique class of asynaptic neurons, the electromotor neurons, whose axons constitute the electric organ. The cell bodies of origin of the electrocyte axons are located in the spinal cord. Both spinal cord and electromotor neurons ( electrocytes ) regenerate after amputation of the tail. Sternarchus spinal cords which have regenerated for 1 or more years show a progression in number of perikarya of electromotor neurons along the rostro-caudal axis. The most recently regenerated region of the cord is at the caudal end, which consists of a tube of ependyma. Progressing rostrally along regenerated spinal cord from the caudal end, numerous cells are generated and large numbers of electromotor neurons differentiate. The maximum number of electromotor neurons per transverse section of regenerated cord is five times higher than in normal mature cord. Rostral to this, the number of electromotor neurons decreases gradually to the normal number near the transition zone (the border with unregenerated cord). As the more rostral regenerated cord has presumably had a longer period of regeneration, we conclude that excess numbers of electromotor neurons are generated initially, and that subsequently the number of these neurons is decreased by cell death. This conclusion is supported by the fact that younger regenerates (2-4 months) have larger-than-normal numbers of perikarya of electromotor neurons extending up to the transition zone (Anderson and Waxman , 1981). No evidence of migration or depletion of electromotor neurons from unregenerated cord rostral to the amputation has been observed. Since the axons of the electromotor neurons in Sternarchus do not normally form any synapses, this study provides evidence that factors other than synaptic competition must be responsible for determining cell death during regeneration of these spinal neurons.     

Deciphering proteins and their functions in the regenerating retina

Neurons of the mammalian CNS, including retinal ganglion cells, lack, in contrast to the PNS, the ability to regenerate axons spontaneously after injury. Regeneration of the CNS is extremely complex and involves various molecular factors and cells. Therewith the regenerative process remains an enormous scientific and clinical challenge. This article provides an overview of proteins that play a crucial role in axon regeneration of retinal ganglion cells and their underlying signaling pathways. In this context, we elucidate the role of 2D gel electrophoresis and highlight some additional proteins, altered upon regeneration by using this highly sensitive method.     

Deciphering proteins and their functions in the regenerating retina

Neurons of the mammalian CNS, including retinal ganglion cells, lack, in contrast to the PNS, the ability to regenerate axons spontaneously after injury. Regeneration of the CNS is extremely complex and involves various molecular factors and cells. Therewith the regenerative process remains an enormous scientific and clinical challenge. This article provides an overview of proteins that play a crucial role in axon regeneration of retinal ganglion cells and their underlying signaling pathways. In this context, we elucidate the role of 2D gel electrophoresis and highlight some additional proteins, altered upon regeneration by using this highly sensitive method.