Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis.

Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1-Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin's GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.

Development of fibroblast-type-II cell communications in fetal rabbit lung organ culture.

The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.

Direct catecholaminergic innervation of spinal dorsal horn neurons with axons ascending the dorsal columns in cat.

Previous ultrastructural studies have shown that catecholamine-containing nerve terminals in the spinal dorsal horn form synaptic junctions with dendrites and somata, but the identity of the neurons giving rise to these structures is largely unknown. In this study we have investigated the possibility that spinomedullary neurons, which project through the dorsal columns to the dorsal column nuclei, are synaptic targets for descending catecholaminergic axons. Neurons with axons ascending the dorsal columns were retrogradely labelled after uptake of horseradish peroxidase by their severed axons in the thoracic (T10-T12) or cervical (C2-C3) dorsal columns. After the retrogradely labelled neurons were visualized, the tissue was immunocytochemically stained with antisera raised against tyrosine hydroxylase or dopamine-beta-hydroxylase. Three hundred forty-three retrogradely labelled neurons within laminae III-V of the lumbosacral dorsal horn were examined under high power with the light microscope. In Triton X-100 treated material, over 60% of cells were found to have dopamine-beta-hydroxylase-immunoreactive varicosities closely apposed to their somata and proximal dendrites. The number of contacts per cell varied from 1 to 22, with a mean number of 4.5. Fewer cells (34%) received contacts from axons immunoreactive for tyrosine hydroxylase as a consequence of the weaker immunoreaction produced by this antiserum. Correlated light and electron microscopic analysis confirmed that many of these contacts were regions of synaptic specialization and that immunostained boutons contained pleomorphic (round to oval) agranular vesicles together with several dense core vesicles. These observations suggest that catecholamines regulate sensory transmission through this spinomedullary pathway by a direct postsynaptic action upon its cells of origin. Such an action would be predicted to suppress transmission generally through this pathway.

Catecholaminergic innervation of the spinal dorsal horn: a correlated light and electron microscopic analysis of tyrosine hydroxylase-immunoreactive fibres in the cat.

The ultrastructural organization of presumed catecholamine-containing boutons, in the dorsal horn of the cat lumbosacral spinal cord, was examined in an immunocytochemical study using an antiserum against tyrosine hydroxylase. The study was restricted to the first four laminae of Rexed. Light microscopic inspection revealed numerous, varicose, tyrosine hydroxylase-immunoreactive axons throughout this region of the spinal cord. Within laminae I and II the fibres exhibited a prominent rostrocaudal orientation, while in laminae III and IV they were organized predominantly dorsoventrally. Correlated ultrastructural analysis confirmed that these varicosities were synaptic boutons. Forty-five of these structures were examined through serial sections and they were found to form symmetrical (Gray type II) synaptic junctions with dendrites (95%) and somata (5%). Immunoreactive boutons were not observed to be either presynaptic or postsynaptic to axon terminals. These findings suggest that catecholamines within the spinal dorsal horn act through a postsynaptic action upon dorsal horn neurons.

Catecholaminergic innervation of the lateral cervical nucleus: a correlated light and electron microscopic analysis of tyrosine hydroxylase-immunoreactive axons in the cat.

The organization of catecholamine-containing axons in the cat lateral cervical nucleus was examined by immunocytochemical methods using a specific tyrosine hydroxylase antiserum. Light microscopic examination revealed numerous tyrosine hydroxylase-immunoreactive axons and varicosities throughout this nucleus, and some of these structures were found in contact with neuronal cell bodies. Correlated ultrastructural analysis showed that these varicosities were synaptic boutons which formed symmetric synaptic junctions with dendrites and somata. This evidence suggests that catecholamines exert a postsynaptic action upon neurons within the lateral cervical nucleus.

Light- and electron-microscopic analysis of neuropeptide Y-immunoreactive profiles in the cat spinal dorsal horn.

The organization of neuropeptide Y-containing profiles in the dorsal horn of cat lumbosacral spinal cord was examined in an immunocytochemical study employing a specific antiserum against neuropeptide Y. Light-microscopic inspection revealed heavy concentrations of immunoreactive axons and varicosities within the superficial layers of the dorsal horn (laminae I and II) and only low to moderate numbers of positive terminals in the deeper layers (laminae III-VI). Neuropeptide-Y immunoreactivity in the superficial laminae occurred primarily as single punctate terminals, although in sagittal sections long rostrocaudally orientated fibres were also found. Immunoreactive fibres in the deeper layers were usually long and beaded. Two-hundred and eight neuropeptide Y-immunoreactive profiles throughout laminae I-VI were examined through serial sections with the electron microscope, and the overwhelming majority (n = 194) was confirmed to be axon terminals, most of which (95%) formed synaptic junctions. These terminals were packed with small irregularly shaped agranular vesicles, together with a number of large dense-core vesicles. Immunoreactivity was homogeneously scattered throughout the cytoplasm, and was also associated with the dense-core vesicles. A few neuropeptide Y-containing profiles (n = 14) were difficult to classify but they could have been vesicle-containing dendrites. The postsynaptic targets of neuropeptide Y-positive terminals were similar throughout each dorsal horn lamina. Most frequently, neuropeptide Y-positive boutons formed axodendritic and axosomatic synaptic junctions (range = 64% of synapses in laminae V/VI to 83% in lamina III). A smaller proportion of synapses were found upon other axon terminals and in laminae I-III the postsynaptic axon terminals were sometimes the central boutons of glomeruli. A number of terminals, especially those in lamina II, formed multiple synapses which often comprised a triadic arrangement. These findings suggest that neuropeptide Y regulates spinal sensory transmission through both a postsynaptic action upon dorsal horn neurons and a presynaptic action upon primary afferent terminals.

Localization of neuronal and endothelial nitric oxide synthase isoforms in human hippocampus.

The aim of the study was to use immunohistochemistry to identify, in the hippocampal region of human brain. the distribution of neuronal and endothelial isoforms of the enzyme nitric oxide synthase. Numerous pyramidal neurons and small, presumed GABAergic interneurons throughout the pyramidal cell layer of CA1-CA3 exhibited neuronal nitric oxide synthase-like immunoreactivity. Comparable immunopositive cells were seen in the granule cell and polymorphic layers of the dentate gyrus and in the stratum oriens. A dense plexus of immunopositive fibres was seen in the granule cell layer of the dentate gyrus. In contrast, endothelial nitric oxide synthase-like immunoreactivity was localized specifically, and with a pronounced punctate distribution, to the cell bodies of CA1 pyramidal neurons. The endothelial isoform was also present in blood vessels and in cells which resembled astroglia. These latter cells had a similar appearance and distribution to astroglia identified by their positive reaction to glial fibrillary acidic protein. The most frequently used method for identifying nitric oxide synthase-containing cells in brain, the NADPH-diaphorase reaction, was also applied to hippocampal sections. Only occasional NADPH-diaphorase-positive cells were seen in the hippocampus where, in contrast to their nitric oxide synthase-like immunoreactivity, the pyramidal cells did not stain for NADPH-diaphorase. Similarly, only occasional NADPH-diaphorase-reactive varicose axons were found in the hippocampus in these experiments. This study is the first to identify mostly separate populations of cells containing neuronal and endothelial nitric oxide synthase isoforms in human hippocampus. The data show that NADPH-diaphorase histochemistry, which is frequently used to show the presence of nitric oxide synthase, greatly underestimates the potential for hippocampal cells to produce nitric oxide. The fact that human hippocampus has a great many nitric oxide synthase-containing cells implies that nitric oxide has a role in human hippocampal functions although, at the present time, these actions are not clear. Whether those stimuli known to produce nitric oxide, such as activation of glutamate N-methyl-D-aspartate receptors, cause both enzyme isoforms in CA1 pyramidal cells to produce nitric oxide remains to be determined.

Substance P receptor (neurokinin-1)-expressing neurons in lamina I of the spinal cord encode for the intensity of noxious stimulation: a c-Fos study in rat.

The substance P receptor neurokinin-1 is expressed by a subset of neurons in the rat spinal cord. We have combined immunostaining for Fos, a marker of noxious peripheral stimulation, and neurokinin-1 to examine whether nociceptive signals from particular peripheral tissues (skin, muscle or knee joint) or activity generated by nerve injury or formalin-induced inflammation are preferentially modulated by substance P. Our results indicate that superficial and deep spinal neurokinin-1-positive neurons process nociceptive information in markedly different ways. In lamina I, the number of double-labelled neurons was positively correlated with the intensity of the stimulus (defined by the total Fos count) and was not directly related to any particular peripheral target. However, in the deeper layers of the spinal cord (V-X), there was no such correlation, and stimulation of joint nociceptors and formalin-induced inflammation produced the greatest proportion of Fos/neurokinin-1 co-localization, suggesting a particular role for substance P in the mediation of joint pain and inflammatory hyperalgesia. Thus, lamina I neurokinin-1 receptor-bearing neurons appear to be involved in intensity discriminative aspects of pain, whereas the deep neurokinin-1 cells are involved in spatial localization or the detection of particular nociceptive submodalities.

A role for spinal lamina I neurokinin-1-positive neurons in cold thermoreception in the rat.

Lamina I neurons of the spinal cord convey specific nociceptive activity to the brain. A subpopulation of lamina I cells bears substance P receptors (neurokinin-1) and recent studies have shown that these neurons encode for the intensity of noxious peripheral stimulation. Here, we report that cool thermal stimuli, applied to the hindpaw of anaesthetized rats, induce Fos expression in lamina I neurokinin-1 neurons that is graded with respect to the intensity of the thermal stimulus. Thus, as the temperature of the stimulus was reduced, both the total number of neurokinin-l-positive neurons expressing Fos and the proportion of Fos nuclei present within neurokinin-1 cells showed a significant increase. These data show that lamina I neurokinin-1 cells encode the intensity of noxious cooling of the skin. In laminae III and IV, although there was no correlation between neurokinin-1 cell activation and stimulus intensity, the total Fos count in these layers was inversely related to the depth of cooling. Thus, neurons in laminae III and IV may also play a role in thermoreception.

Low-frequency stimulation induces homosynaptic depotentiation but not long-term depression of synaptic transmission in the adult anaesthetized and awake rat hippocampus in vivo.

The induction of homosynaptic long-term depression and depotentiation of previously established long-term potentiation was investigated in the CA1 hippocampal region of anaesthetized and awake adult rats following prolonged ipsilateral low-frequency stimulation of the Schaffer collateral/ commissural pathway. Prolonged low-frequency stimulation at 1-10 Hz failed to induce long-term depression of field excitatory postsynaptic potentials in the anaesthetized or awake adult rat. However, prolonged low-frequency stimulation at 5 and 10 Hz, although not at 1 or 2 Hz, did induce depotentiation of previously established long-term potentiation in anaesthetized animals. Thus, in the anaesthetized animals, 900 pulses at 10 Hz induced a depotentiation of 68%, 59% and 66% when given 10, 30 and 40 min following long-term potentiation induction. Depotentiation could also be induced at much longer times following the induction of long-term potentiation. Thus, in anaesthetized rats, depotentiation measuring 34% was induced by 10-Hz stimulation 4 h following long-term potentiation induction, and depotentiation measuring 60% was induced in two sets of experiments 24 h after long-term potentiation induction in awake animals. The results of the present study show that homosynaptic long-term depression was not induced in the adult hippocampus in vivo using stimulation protocols which are effective in hippocampal slices. However, erasure of long-term potentiation by the process of depotentiation has been shown to occur in the adult hippocampus in vivo, both at short times and at prolonged times after the induction of long-term potentiation.

A prospective study of asthma in a rural community.

Changes in symptoms and pulmonary function among asthmatic subjects in the general population remain poorly characterized. We studied 1,303 white residents aged seven years and older in Lebanon, Conn, a rural community largely unaffected by air pollution or major occupational exposures. These residents were examined in 1972 and again in 1978. There were 73 asthmatic subjects seen in 1972 who were followed. In addition, we identified 278 persons in 1972 who complained of wheezing who were also seen in 1978. Of the original asthmatic subjects, 50 (68 percent) were in remission; and from the original nonasthmatic population, 19 (1.4 percent) new asthmatic subjects were identified. Similarly, the condition of 215 (77 percent) of those who initially complained of wheeze had improved, whereas 56 (4.6 percent) of those initially studied either developed new wheeze or saw their wheezing worsen. When the groups of persons complaining of wheeze and the asthmatic subjects were analyzed for the presence of chronic bronchitis, we found a significant correlation between wheeze and chronic bronchitis in individuals aged 18 years and older (p less than 0.001) for both men and women, and a significant correlation (p less than 0.001) between asthma and chronic bronchitis in women aged 18 years and older. Loss of pulmonary function over time measured in terms of the forced expiratory volume in one second and the forced expiratory flow at 50 percent of total lung capacity was consistently greater for asthmatic adults than for nonasthmatic adults. Furthermore, when individuals were studied by the severity and duration of their asthmatic symptoms, a trend of worse pulmonary function was seen in those individuals with chronic asthma. We conclude that remission rates among asthmatic subjects and persons with wheeze are high in individuals aged seven years and older, that chronic bronchitis is frequently associated with wheezing and a history of asthma in adults, and that significant abnormalities in pulmonary function as well as accelerated loss of pulmonary function are associated with asthma.

Reduced nuclear factor kappaB (p65) expression in rat primary sensory neurons after peripheral nerve injury.

The activated form (p65) of nuclear factor kappa B (NF-kappa B) was found to be expressed in a sub-population (32%) of mixed diameter rat sensory neurons in L4 and L5 dorsal root ganglia, indicating that this transcription factor is involved in intracellular signalling in sensory neurons under physiological conditions. Four hours after crushing the sciatic nerve, ipsilateral p65 staining was abolished in a subgroup (60-70%) of these neurons. The contralateral side was unaffected by the injury and loss of NF-kappa B activity was not observed following sham surgery. Within 24 h of sciatic injury, ipsilateral p65 staining had recovered to control levels. We suggest that the decline in p65 after nerve injury was due to failure of retrograde axonal transport of trophic factor(s). Since neurotrophins and cytokines promote the survival of non-neuronal cells by activation of NF-kappa B, we believe that p65 may be critical for the resistance to apoptosis shown by adult sensory neurons.

Cefprozil versus cefaclor in the treatment of acute and uncomplicated urinary tract infections. Cefprozil Multicenter Study Group.

Cefprozil is a new oral cephalosporin with an in vitro spectrum of activity that includes the pathogens most commonly associated with acute and uncomplicated urinary tract infections (UTIs). A multicenter, randomized study was conducted to compare the clinical efficacy and safety of cefprozil, administered once daily, with cefaclor, administered three times a day, for ten days in patients 2 years of age or older who had acute and uncomplicated UTIs. The rate of satisfactory clinical response in evaluable patients was 87% in the cefprozil group and 84% in the cefaclor group. The patient bacteriologic response rates were also similar: 83% for cefprozil and 85% for cefaclor. The overall effective response rate for both cefprozil and cefaclor was 77%. Both drugs were well tolerated, with no difference in the incidence of drug-related adverse events. Because of its efficacy and once-daily dosing regimen, cefprozil may be an alternative to currently available oral antibiotics in the treatment of UTIs.

Cefprozil versus cefaclor in the treatment of mild to moderate skin and skin-structure infections. The Cefprozil Multicenter Study Group.

In a multicenter study, 598 patients with skin or skin-structure infections were randomly assigned to receive 500 mg of cefprozil once daily (or 20 mg/kg once daily) or 250 mg of cefaclor three times daily (or 20 mg/kg daily in three equal doses) for 5 to 10 days. Treatment was evaluated in 212 cefprozil-treated patients and in 210 cefaclor-treated patients. The patients were aged 2 to 99 years (mean, 28 years) and their primary diagnoses were impetigo (in 99 patients), pyoderma (in 98), superficial abscess (in 70), and cellulitis (in 64). A satisfactory clinical response was found in 93% of the cefprozil-treated patients and in 92% of the cefaclor-treated patients, the pathogens were eradicated in 91% and 89%, and overall treatment was rated effective in 87% of both groups. Adverse clinical events were reported by 5% of the patients in both groups; one cefprozil-treated patient and three cefaclor-treated patients withdrew from treatment because of adverse events. It is concluded that cefprozil administered once daily is as effective and safe as cefaclor administered three times daily in the treatment of mild to moderate skin and skin-structure infections.

Ultrastructural analysis of noradrenergic nerve terminals in the cat lumbosacral spinal dorsal horn: a dopamine-beta-hydroxylase immunocytochemical study.

Noradrenaline-containing nerve terminals within the cat spinal dorsal horn were studied by immunocytochemical localization of dopamine-beta-hydroxylase. Immunoreactive terminals formed symmetrical (Gray type II) synaptic specializations with dendrites and somata throughout laminae I-IV, but no junctions were formed with other axons. These findings suggest that noradrenaline regulates sensory transmission through the dorsal horn via a postsynaptic action.

Neuropeptide Y-immunoreactive terminals form axo-axonic synaptic arrangements in the substantia gelatinosa (lamina II) of the cat spinal dorsal horn.

The ultrastructural organization of nerve terminals containing neuropeptide Y-immunoreactivity was studied in the substantia gelatinosa of the cat spinal dorsal horn. Seventy immunoreactive boutons were examined through serial sections and 67 of them were found to form between one and five synaptic junctions with dendrites (59.5% of synapses), somata (3% of synapses) and other axon terminals (37.5% of synapses). The postsynaptic axon terminals were often the central boutons of glomeruli. These findings suggest that neuropeptide Y regulates spinal sensory transmission through both a postsynaptic action upon dorsal horn neurons and a presynaptic action upon primary afferent terminals.

Relationships between spinocervical tract neurons and descending catecholamine-containing axons in the cat.

Lumbosacral (L6-S1) spinal cord neurons in the cat were retrogradely labelled after uptake of horseradish peroxidase by their severed axons in the upper cervical (C3-C4) dorsolateral funiculus. Sections of L6-S1 containing labelled neurons were then processed immunocytochemically using antibodies against dopamine-beta-hydroxylase or tyrosine hydroxylase, two enzymes responsible for the synthesis of catecholamines. Two hundred and ninety eight retrogradely-labelled cells within laminae III-V of the dorsal horn were examined under high power (x 1000) with the light microscope. In Triton X-100-treated material, only 13% of these cells had catecholamine-containing varicosities closely apposed to their somata and proximal dendrites, which suggests that in comparison with the postsynaptic dorsal column pathway, spinocervical tract neurons are only sparsely innervated by descending catecholaminergic axons.

Application of [3H]L-N(G)-nitro-arginine labelling to measure cerebellar nitric oxide synthase in patients with schizophrenia.

Brains from patients with schizophrenia have been reported to contain deficient and dysplastic forebrain neurons containing nitric oxide synthase (NOS). As part of a study of NOS in schizophrenia, we decided to investigate the cerebellum, which has particularly high levels of NOS. We used an autoradiographic method to measure the density and distribution of NOS. Sections of frozen cerebellum removed at autopsy were labelled with the selective NOS inhibitor [3H]L-NG-nitro-arginine. NOS levels were visualized in sagittal sections of vermis from 16 control subjects and 21 schizophrenia patients, and measurements were taken from the three groups of developmentally-distinct lobules I-V, VI-VII and VIII-X. The highest NOS density was in the Purkinje/molecular layer of cerebellar cortex, although there was some NOS in the granule cell layer. There were no differences in Purkinje/molecular or granule cell layer NOS levels between the two groups of subjects. The mild structural faults in cerebellar vermis observed in some patients with schizophrenia probably do not involve reductions in NOS-containing cells.

Use of MRI for measuring structures in frozen postmortem brain.

A method was developed for magnetic resonance imaging (MRI) of human autopsy brains stored long-term at -70 degrees C. Scanning brains at temperatures between -70 and -8 degrees C gave minimal MRI signals consistent with protons having limited freedom of movement at low temperature. Raising brain temperature improved the signal such that scanning at -1 degree C generated images with good in-plane resolution, grey/white matter contrast, and fine detail of cortical sulcal/gyral patterns. To validate the method, volume and area measurements were made using computerized image analysis on stored digital images of 14 brains from adult subjects of both genders and various ages. The data confirmed that brain volume was inversely correlated with age, and female subjects had smaller brains. This is a valuable new method for acquiring morphometric data from previously unscanned pathologic brains that are to be used for neurochemical and molecular investigations.

Characterization of liquid chromatographic stationary phases by Raman spectroscopy. Effect of ligand type.

This study represents the first Raman spectroscopic characterization of conventional chemically-bonded liquid chromatographic (LC) stationary phases under typical flow-rate and pressure conditions. Raman spectra were obtained for amino propyl (NH2), cyano propyl (CN), phenyl (Ph), octadecyl (C18), octyl (C8), and methyl (C1) chemically-bonded silica-based stationary phases in 100% aqueous mobile phases. The present experimental set-up has allowed Raman spectra of various stationary phase ligands, present in sub-monolayer coverages on the siliceous supports, to be obtained. This study: (1) demonstrates that conventional Raman spectroscopic techniques can be used to study LC stationary phases; (2) presents the experimental set-up, conditions, and approaches utilized to obtain Raman spectra of conventional stationary phases; (3) examines the spectroscopic differences observed for a variety of different types of bonded ligands that are typically used in reversed-phase (RPLC) and normal-phase (NPLC) liquid chromatographic separations; and (4) considers other future studies that are possible with this experimental approach, including mobile phase composition and temperature studies.