Binding, internalization, and membrane incorporation of human immunodeficiency virus-1 at the blood-brain barrier is differentially regulated.

Human immunodeficiency virus (HIV)-1 within the CNS induces neuro-acquired immunodeficiency syndrome and acts as a reservoir for reinfection of peripheral tissues. HIV-1 crosses the blood-brain barrier (BBB) within infected immune cells and as cell-free virus by a CD4-independent mechanism. Which proteins control free virus transport across the BBB are unknown, but work with wheatgerm agglutinin (WGA) and heparin suggests that heparan sulfate proteoglycans, sialic acid, and N-acetyl-beta-D-glucosaminyl acid bind HIV-1. Here, we found that an HIV-1 T-tropic virus was taken up by mouse brain endothelial cells in vitro and crossed the BBB in vivo and could be effluxed as intact virus. Uptake was stimulated by WGA and protamine sulfate (PS) and inhibited by heparin. BBB uptake of virus involved four distinguishable binding sites: i) reversible cell surface binding involving gp120 and sensitive to PS/heparin but insensitive to WGA; internalization with a ii) WGA-sensitive site binding gp120 and iii) a PS/heparin-sensitive site not involving gp120; iv) membrane incorporation not affected by WGA, heparin, or PS. In conclusion, binding, internalization, and membrane incorporation are separately regulated steps likely determining whether HIV-1 is incorporated into brain endothelial cells, transported across them, or returned to the circulation.

Transport of human immunodeficiency virus type 1 pseudoviruses across the blood-brain barrier: role of envelope proteins and adsorptive endocytosis.

Blood-borne human immunodeficiency virus type 1 (HIV-1) crosses the blood-brain barrier (BBB) to induce brain dysfunction. How HIV-1 crosses the BBB is unclear. Most work has focused on the ability of infected immune cells to cross the BBB, with less attention devoted to the study of free virus. Since the HIV-1 coat glycoprotein gp120 can cross the BBB, we postulated that gp120 might be key in determining whether free virus can cross the BBB. We used radioactive virions which do (Env+) or do not (Env-) bear the envelope proteins to characterize the ability of HIV-1 to be taken up by the murine BBB. In vivo and in vitro studies showed that the envelope proteins are key to the uptake of free virus and that uptake was enhanced by wheat germ agglutinin, strongly suggesting that the envelope proteins induce viral adsorptive endocytosis and transcytosis in brain endothelia. Capillary depletion showed that Env+ virus completely crossed the vascular BBB to enter the parenchyma of the brain. Virus also entered the cerebrospinal fluid, suggesting passage across the choroid plexus as well. About 0.22% of the intravenously injected dose was taken up per g of brain. In vitro studies showed that postinternalization membrane cohesion (membrane binding not reversed with acid wash or cell lysis) was a regulated event. Intact virus was recovered from the brain endothelial cytosol and was effluxed from the endothelial cells. These results show that free HIV-1 can cross the BBB by an event related to adsorptive endocytosis and mediated by the envelope proteins.

Fiberoptic bronchoscopic cryotherapy in the management of tracheobronchial obstruction.

Cryotherapy is used for endoscopic management of tracheobronchial obstruction (TBO). This study describes the use of a flexible cryoprobe for cryotherapy using nitrous oxide as a cryogen through a fiberoptic bronchoscope. The study group consisted of 22 patients, ages ranging from 28 to 82 years. Twenty patients had malignant TBO and two had bronchial obstruction (BO) following lung transplantation. Benign BO was first dilated with a balloon and followed with cryotherapy. Eighteen of the 20 malignant endobronchial lesions were completely removed. In three of these patients, the airway remained occluded due to extrinsic compression. Cryotherapy offers an alternative to Nd:YAG laser in the management of TBO. Cryotherapy offers other advantages such as being inexpensive, safe for the operator, and safe for other members of the team. Similarly for the patient, there is no danger of bronchial wall perforation or endobronchial fires, cryotherapy can be done under local anesthesia with conscious sedation, and it can be performed in an endoscopy suite.

Two novel alpha-neurotoxins isolated from the taipan snake, Oxyuranus scutellatus, exhibit reduced affinity for nicotinic acetylcholine receptors in brain and skeletal muscle.

Three novel toxic peptides were purified to homogeneity from the venom of the Australian taipan snake, Oxyuranus scutellatus scutellatus. On the basis of complete amino acid sequence analyses, two of these toxins belong to the family of short-chain alpha-neurotoxins found in elapid and hydrophid snake venoms and are the first postsynaptic neurotoxins identified in taipan venom. Radioligand binding studies confirm that taipan toxins 1 and 2 inhibit the binding of [125I]-alpha-bungarotoxin to nicotinic acetylcholine receptors in skeletal muscle with IC50 values of 2.4-2.5 nM but are 5-fold less potent in this assay than alpha-bungarotoxin or the two short-chain alpha-neurotoxins erabutoxin a and erabutoxin b. Taipan toxins 1 and 2 do not antagonize [125I]-alpha-bungarotoxin binding to central neuronal nicotinic receptors at concentrations up to 3 microM. We find that erabutoxin a and erabutoxin b do inhibit the binding of [125I]-alpha-bungarotoxin to central neuronal nicotinic receptors but are over 350-fold less potent than long-chain alpha-neurotoxins at these receptors. The novel alpha-neurotoxins from taipan venom do not inhibit the binding of [3H]nicotine to high-affinity nicotine receptors in brain, a property they share with alpha-bungarotoxin and the erabutoxins. The results demonstrate that at least two neuromuscular junction-blocking peptides are present in taipan venom. Nonconservative substitutions at position 32 in both taipan toxin 1 and 2 may be responsible for the observed decreases in affinities of the toxins of 5-fold for muscle receptors (compared to alpha-bungarotoxin) and over 10-fold for alpha-bungarotoxin-sensitive nicotinic receptors in brain (compared to the structurally similar short-chain alpha-neurotoxins erabutoxin a and erabutoxin b).

Nicotinic acetylcholine receptors in separate brain regions exhibit different affinities for methyllycaconitine.

The family of nicotinic acetylcholine receptors contains numerous subtypes. Since the subunit compositions of most native neuronal nicotinic receptors are unknown, an important method for distinguishing subtypes of functional neuronal receptors is based on pharmacological criteria, such as affinity for snake toxins. We have now examined the affinities of native chick nicotinic receptors for methyllycaconitine, a toxin purified from Delphinium. We find that methyllycaconitine is a potent antagonist at central nicotinic receptors located on Edinger-Westphal neurons, producing nearly complete functional blockade of nicotinic responses at 10 nM. In marked contrast, methyllycaconitine is 1000-fold less potent at blocking nicotinic responses in the lateral spiriform nucleus. Methyllycaconitine inhibits kappa-bungarotoxin-sensitive nicotinic receptors in ciliary ganglia at 0.5-1.0 microM. Radioligand binding studies also reveal heterogeneity in the affinity of the toxin for nicotinic receptors. Methyllycaconitine binds most avidly to [125I] alpha-bungarotoxin sites in brain (Ki = 5.4 nM), and is 200-fold less potent at muscle nicotinic receptors (IC50 = 1.1 microM). The least potent binding of the toxin is to [3H]nicotine sites in brain (Ki = 3.7 microM). Methyllycaconitine is thus a useful pharmacological tool for distinguishing certain subtypes of native nicotinic receptors. The relatively low affinity of the toxin for nicotinic receptors in the lateral spiriform nucleus is consistent with the known properties of these receptors, which include a high affinity for [3H]nicotine and a lack of sensitivity to alpha- and kappa-bungarotoxin. On the basis of high affinity for methyllycaconitine and insensitivity to alpha-bungarotoxin, the nicotinic receptors in the Edinger-Westphal nucleus are unlike any previously described nicotinic receptor subtype.

Management of postpneumonectomy syndrome with a bronchoscopically placed endobronchial stent.

Postpneumonectomy syndrome is a rare complication of right pneumonectomy characterized by marked shift and counterclockwise rotation of the heart and mediastinum into the evacuated hemithorax. Typically the left primary bronchus is draped over the aorta or spine and is markedly compressed. We describe one patient with postpneumonectomy syndrome successfully managed nonsurgically with a bronchoscopically placed Silastic endobronchial stent.

Functional heterogeneity of nicotinic receptors in the avian lateral spiriform nucleus detected with trimethaphan.

We have examined an excitatory response mediated by nicotinic acetylcholine receptors located on the somata and/or dendrites of chick lateral spiriform neurons. On the basis of pharmacological and anatomical studies, these receptors belong to a subgroup of nicotinic receptors termed high affinity nicotine receptors, because they exhibit a high affinity for nicotinic agonists but little or no sensitivity to alpha- or kappa-bungarotoxin. We now report physiological evidence that high affinity nicotine receptors in the lateral spiriform nucleus are heterogeneous. Intracellular recording in brain slices was used to examine the pharmacological characteristics of nicotinic responses in individual lateral spiriform neurons. Nicotinic responses to brief applications of carbachol were inhibited by trimethaphan, dihydro-beta-erythroidine, or d-tubocurarine. Trimethaphan was unusual, in that a wide range of concentrations (< or = 50 microM to > 500 microM) were required to block this nicotinic response in different neurons. To quantitate the inhibition observed with trimethaphan and dihydro-beta-erythroidine, dose-response curves were generated in superfusion studies using a wide range of concentrations of both agonist (3-3000 microM carbachol in the presence of 1 microM atropine and 0.25 microM tetrodotoxin) and antagonists (10-500 microM trimethaphan or 0.1-3 microM dihydro-beta-erythroidine). The data yielded an EC50 of 25 +/- 5 microM for carbachol, with a Hill coefficient of 1.4 +/- 0.1 (mean +/- standard error; n = 8). In the case of dihydro-beta-erythroidine, a narrow range of Ki values was obtained (0.09-0.16 microM; n = 5). In contrast, Ki values for trimethaphan varied over a 15-fold concentration range (4-66 microM; n = 17), demonstrating that trimethaphan showed selectivity for different receptor subtypes found in the lateral spiriform nucleus. For both antagonists, the data indicate a competitive mode of inhibition.

Symptomatology, pulmonary function and response, and T lymphocyte beta 2-receptors during smoking cessation in patients with chronic obstructive pulmonary disease.

STUDY OBJECTIVES: To characterize the effect of smoking cessation and nicotine replacement on pulmonary symptomatology, baseline pulmonary function and response to terbutaline, and purified T lymphocyte beta 2-receptor regulation; and the relationship between T lymphocyte beta 2-receptor density and pulmonary function. DESIGN: Open-label, longitudinal, 28-week study. SETTING: A university clinical research center. PATIENTS: Eighteen long-term smokers with mild to moderate chronic obstructive pulmonary disease (COPD) were enrolled and seven completed the study. INTERVENTIONS: Subjects stopped smoking with the aid of nicotine substitution and behavioral counseling. Pulmonary response (FEV1) to subcutaneous terbutaline and T lymphocyte beta 2-receptor density (Bmax) and function (cAMP) were measured prior to smoking cessation (week 0), during nicotine replacement (week 8), and after nicotine cessation (week 28). MEASUREMENTS AND MAIN RESULTS: Serum cotinine concentrations, plasma epinephrine concentrations, and day and night cough decreased significantly after smoking cessation, whereas basal cAMP concentrations increased (p < 0.05). No significant change was seen in baseline FEV1, pulmonary response to terbutaline, or Bmax over the 28 weeks; however, intrasubject changes in Bmax between visits correlated significantly (p < 0.05) with intrasubject changes in pulmonary response between visits. CONCLUSIONS: Our data indicate that smoking cessation is associated with a significant decrease in the symptomatology of COPD, and that change in T lymphocyte beta 2-receptor density is a good marker of change in pulmonary response to beta 2-agonists.

Different responses to opioids measured in terminals and somas of Edinger-Westphal neurons.

Neurotransmitter receptors on the axon terminals of a neuron can be located a considerable distance away from comparable receptors on the cell body or dendrites of the same neuron. We examined the effects of activating either nerve terminal receptors or those located on or near the somas of chick Edinger-Westphal neurons. Cell body responses were measured via intracellular recording in a brain slice preparation. To measure nerve terminal responses, intracellular recordings were obtained from the large, calyciform nerve endings in intact ciliary ganglia, which emanate from neurons of the lateral Edinger-Westphal nucleus. Cell bodies of Edinger-Westphal neurons responded to leucine-enkephalin with a dose-dependent hyperpolarization that was associated with a decrease in input resistance. In spontaneously active Edinger-Westphal somas, leucine-enkephalin caused marked inhibition of suprathreshold and subthreshold activity, indicating that, as with a number of other central neurons, the major effect of opioids was to reduce excitability. The response to opioids was sensitive to naloxone (1 microM) and was a direct effect, since it was not blocked by either 0.5 microM tetrodotoxin or 100 microM cadmium. More selective mu ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin) and delta ([D-Ser2]-leucine-enkephalin-Thr and [D-Pen2,5]-enkephalin) opioid agonists produced effects similar to those of leucine-enkephalin. Opioids produced strikingly different effects in the nerve terminals of Edinger-Westphal neurons, where the major effect was a depolarization associated with a decrease in input resistance. The effects of opioids in the terminals were reduced in a low sodium buffer, indicating that they were dependent on the presence of extracellular sodium.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged delirium tremens requiring massive doses of medication.

Delirium tremens might last for weeks and treatment requires massive benzodiazepine doses, yet it is possible to manage patients with this condition successfully. In this case of delirium tremens, standard agents at the usual recommended doses were not sufficient to achieve control of confusion and agitation or to stabilize neurologic and cardiovascular parameters. The patient required extraordinarily high doses of central nervous system depressants for an extended period. Midazolam, a short-acting benzodiazepine, was used but was associated with metabolic acidosis and was extremely expensive. Although high-dose midazolam should probably be avoided, extremely high dose benzodiazepine use for an extended period might be necessary in some cases. In this circumstance we advise diazepam because of its low cost and relative safety. The tendencies to withhold large doses for fear of side effects or to give up in cases requiring prolonged intensive support must be resisted to minimize the mortality from this severe illness.

In vivo and in vitro beta 2-adrenergic receptor responsiveness in young and elderly asthmatics.

This pilot study was undertaken to characterize age-related alterations in airway and metabolic beta 2-adrenergic receptor response using terbutaline as a probe. A single dose of terbutaline 0.007 mg/kg was administered subcutaneously to 10 young (range 20-35 yrs) and 10 elderly (range 60-73 yrs) men and women with asthma. beta-Receptor function was assessed by serial pulmonary function tests, heart rate, blood pressure, plasma potassium, glucose, insulin, and catecholamine levels, and in vitro 3', 5'-cyclic adenosine monophosphate (cAMP) production of pure T lymphocytes. The trend was for lung beta 2-receptor response (bronchodilation) and T lymphocyte cAMP production to be lower in elderly than in young asthmatics, but differences did not reach statistical significance. Elderly subjects' beta 1-adrenergic receptor responsiveness (systolic blood pressure) was also dampened. These data suggest that T lymphocyte stimulation does reflect pulmonary beta 2-receptor response. Whether elderly asthmatics require and tolerate higher dosages of parenteral terbutaline than younger asthmatics deserves further study.

[Effect of temperature on the growth of Penicillium from moldy feed]. / Einfluss der Temperatur auf das Wachstum von Penicillien aus verschimmelten Futtermitteln.

178 Penicillium strains were isolated from moulded feedstuffs (mainly corn and wheat after ambient air drying, wheat after refrigeration, corn after treatment with propionic acid), and investigated for the effect of temperature on growth rate. Temperatures between 4 and 27 degrees C were used. 159 strains proved psychrotrophic, they were able to grow at 4 degrees C on malt extract agar (MA) as well as in wheat; the growth in wheat was indicated by the production of ergosterol. On MA all psychrotrophic strains were able to grow also at 10-27 degrees C. The maximum rate of growth was reached at 10-15 degrees C, 15-20 degrees C, 20-27 degrees C, or must be assumed for temperatures greater than or equal to 27 degrees C. At 4 and 10 degrees C the mean growth rate on MA was 24 and 51%, respectively, related to the mean rate at 20 degrees C; the lower temperature limit of growth was found at - 1 degree C by graphic extrapolation. The Q10 value of growth on MA is about 2, if temperatures between 20 and 10 degrees C are compared; with decreasing temperature higher Q10 values up to 3.7 are obtained. The growth rate of a strain of Penicillium aurantiogriseum on wheat was affected by temperature nearly in the same manner as on malt extract agar. The results are discussed with respect to the risk of moulding during refrigerated storage of feedstuffs.

Kappa 2-bungarotoxin and kappa 3-bungarotoxin: two new neuronal nicotinic receptor antagonists isolated from the venom of Bungarus multicinctus.

Neuronal nicotinic acetylcholine receptors are recognized with high affinity by two snake venom kappa-neurotoxins, kappa-bungarotoxin and kappa-flavitoxin. Native and radiolabeled kappa-neurotoxins have been used to localize and quantitate neuronal nicotinic receptors in a variety of species. We now report the identification of two new kappa-neurotoxins. kappa 2-Bungarotoxin and kappa 3-bungarotoxin were purified from the venom of Bungarus multicinctus collected in the province of Guangdong, China. kappa-Bungarotoxin has as yet not been found in this venom, although it is the only kappa-neurotoxin to be isolated thus far from Taiwanese Bungarus multicinctus. The geographical separation of Guangdong and Taiwan might account for this evolutionary divergence within the species. Both of the new kappa-neurotoxins are potent antagonists of nicotinic transmission in the chick ciliary ganglion. kappa 3-Bungarotoxin, the least potent of the kappa-neurotoxins, produces a complete blockage of nicotinic transmission in 60 min at 250 nM. Protection experiments using the short-acting nicotinic antagonists dihydro-beta-erythroidine and (+)-tubocurarine demonstrate that kappa 2-bungarotoxin blocks transmission by binding to the acetylcholine recognition sites of neuronal nicotinic receptors. The isoelectric point of kappa 2-bungarotoxin (pI = 8.9) is similar to that of kappa-bungarotoxin and kappa-flavitoxin, but kappa 3-bungarotoxin is considerably more basic, with pI greater than 11. Partial amino acid sequences are reported for both kappa 2-bungarotoxin and kappa 3-bungarotoxin. These sequences show a high degree of homology (approximately 80%) with other kappa-neurotoxins, and allow the determination of the critical differences between the kappa-neurotoxins and the structurally related alpha-neurotoxins. For example, all 4 kappa-neurotoxins lack a tryptophanyl residue which is invariant and important for function in the alpha-neurotoxins. The kappa-neurotoxins also differ from the alpha-neurotoxins by having an invariant prolinyl residue at a critical sequence position. Heterodimers were detected consisting of one subunit each of kappa 2-bungarotoxin and kappa 3-bungarotoxin. These heterodimers, which form between any combination of two kappa-neurotoxins, appear to be physiologically active and confirm that a further distinction between kappa-neurotoxins and alpha-neurotoxins is the strong tendency of the former to self-associate in solution. The present results help to establish the definition of 'kappa-neurotoxin'. These snake toxins are now being used by a number of laboratories in physiological and biochemical experiments on neuronal nicotinic receptors from a variety of species.(ABSTRACT TRUNCATED AT 400 WORDS)

Kappa-neurotoxins: heterodimer formation between different neuronal nicotinic receptor antagonists.

The kappa-neurotoxins are a family of snake venom polypeptides that are competitive antagonists of acetylcholine at a variety of neuronal nicotinic receptors. We have previously determined that kappa-bungarotoxin, purified from the venom of Bungarus multicinctus, exists in solution entirely as a dimer of identical subunits. We now report that the three other known kappa-neurotoxins, namely, kappa 2-bungarotoxin and kappa 3-bungarotoxin from Bungarus multicinctus and kappa-flavitoxin from Bungarus flaviceps, also self-aggregate in solution. Furthermore, when two different kappa-neurotoxins are mixed, a heterodimer species spontaneously forms and reaches an equilibrium with the two homodimers after which 40-50% of the protein exists as the heterodimer. A cation-exchange high-pressure liquid chromatography procedure is described which readily separates kappa-neurotoxin heterodimers from the homodimers. Sedimentation equilibria experiments give an Mr = 15,500 +/- 1000 for kappa-flavitoxin and an Mr = 14,500 +/- 700 for a mixture of kappa-bungarotoxin and kappa-flavitoxin. Since the subunit molecular weights of kappa-bungarotoxin and kappa-flavitoxin are respectively 7313 and 7242, self-aggregation of these toxins in solution results in a preponderance of kappa-neurotoxin dimers. The stoichiometry of the heterodimer formed by kappa-bungarotoxin and kappa-flavitoxin is 1:1, as determined by amino acid sequence analysis. After isolation, the kappa-neurotoxin heterodimer partially dissociates and again reaches equilibrium with the homodimers, a process which requires 2-4 h at 23 degrees C. The ability to self-aggregate to form heterodimers and homodimers thus appears to be a common property of the kappa-neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucoepidermoid carcinoma of the lung with intracranial metastases.

Discussed is the first roentgenographic and post-mortem description of a patient with mucoepidermoid carcinoma of the lung who presented with intracranial metastases. The patient's primary tumor eluded physical diagnosis and bronchoscopic delineation. The autopsy confirmed minimal tumor involvement of the bronchial wall despite bulky regional and distant metastases.

kappa-Bungarotoxin: binding of a neuronal nicotinic receptor antagonist to chick optic lobe and skeletal muscle.

kappa-Bungarotoxin, a snake venom kappa-neurotoxin, is a potent neuronal nicotinic receptor antagonist. kappa-Neurotoxins are structurally related to the long-type alpha-neurotoxins (including alpha-bungarotoxin), which often fail to block neuronal nicotinic transmission, but which are potent antagonists of nicotinic receptors found on vertebrate skeletal muscle. The binding of kappa-bungarotoxin has now been examined in homogenates of chick skeletal muscle and optic lobe. In muscle, kappa-bungarotoxin binds to nicotinic receptors with 200-fold lower affinity than does alpha-bungarotoxin. The weakest known alpha-neurotoxin, L.s. III, is found to be 6.5-fold more potent than kappa-bungarotoxin. These findings support the conclusion that kappa-neurotoxins are selective for neuronal nicotinic receptors. In the optic lobe, 125I-alpha-bungarotoxin and 125I-L.s. III. A second nicotinic site, detected with high affinity by both alpha-neurotoxins, is only weakly bound by kappa-bungarotoxin. No evidence for a unique 125I-kappa-neurotoxin site is observed. Furthermore, kappa-bungarotoxin does not recognize the high affinity L-[3H]nicotine binding site in chick optic lobe which is distinct from the alpha-neurotoxin binding sites. Three subtypes of nicotinic sites can thus be defined in chick optic lobe, although which of these subtypes is involved in nicotinic transmission in the lobe remains to be conclusively determined.

Malignant mesothelioma with occupational and environmental asbestos exposure in an Illinois community hospital.

Clinical, radiologic, pathologic, and epidemiologic data on 32 patients with diffuse malignant mesothelioma (DMM) diagnosed between 1968 and 1984 at a 427-bed community hospital in Berwyn, Ill, were reviewed. Independent pathologists' review of light microscopy, supported by electron microscopy, immunoperoxidase staining, or autopsy, confirmed 29 pleural and three peritoneal DMMs. Clinical and radiologic characteristics were similar to those in published case series. Median age at diagnosis was 67 years, and median survival after diagnosis, seven months. Fourteen patients were women. Exposure histories were obtained through 22 interviews supplemented by hospital charts and death certificates. Thirty patients (94%) had a history of asbestos exposure through work (15 [47%]) and/or residence near an asbestos facility (27 [84%]). Medical records and death certificates underreported asbestos exposure and DMM.