Gracilis or semitendinosus myopathy in 18 dogs.

The clinical findings in 18 dogs with gracilis (n = 17) or semitendinosus (n = 1) myopathy are described. Each dog had a similar hind-limb gait abnormality characterized by a shortened stride with a rapid, elastic medial rotation of the paw, internal rotation of the hock and external rotation of the calcaneus [corrected] and internal rotation of the stifle during the mid-to-late swing phase of the stride. Medical management prior to or in lieu of surgery was attempted (n = 8) with no apparent response. Fifteen dogs had one or multiple surgical procedures. Although transection, partial excision, or complete resection of the affected muscle resulted in resolution of lameness following surgery, lameness recurred six weeks to five months (mean, 2.5 months; median, two months) following surgery. Adjunctive medical treatment did not prevent recurrence. Variable replacement of the affected muscle with fibrous connective tissue (predominantly along the caudolateral border of the muscle) was evident grossly, and replacement of myofibers with fibrous connective tissue was confirmed histologically. A definitive etiology could not be established.

Characterization of synaptic vesicles and related neuronal features in nerve growth factor and ras oncogene differentiated PC12 cells.

PC12 cells can differentiate into neuron-like cells after treatment with either nerve growth factor (NGF) or transduction with a retrovirus which expresses the K-ras oncogene. The concomitant treatment of NGF plus ras differentiates PC12 cells further than either agent alone with respect to neurite outgrowth, acetylcholinesterase levels, and most strikingly, the number of synaptic vesicle (SV) clusters. These SV clusters in PC12 cell neurites closely resemble those in the presynaptic terminals of neurons. Such SV clusters have not been described in cell lines previously. The SV clusters from all three differentiated groups (NGF, ras, and NGF plus ras) were similar in size, shape, and configuration, except that the ones in the doubly treated group occur in higher frequency and have more vesicles. The synaptic nature of these vesicle clusters was demonstrated by their regulated depletion after potassium stimulation. Furthermore, these vesicle clusters stained positively for two SV-associated proteins, synapsin I and synaptophysin, by EM immunocytochemistry (ICC). Such SV clusters in a cell line are very useful for characterizing the regulated release of SVs and the distribution of SV-related antigens in intact cells. Analysis by SDS-gel electrophoresis and immunoblotting indicated that synapsin I levels are higher in all three differentiated groups compared to untreated cells; whereas synaptophysin levels are lower in cells exposed to NGF alone or with NGF and ras double treatment. Possible convergence and/or divergence on the mechanisms of NGF and ras differentiation in PC12 cells are discussed.

The laryngeal mask airway in infants.

Clinical and fibreoptic assessment of positioning of the size 1 laryngeal mask airway was performed in 50 infants. A clinically patent airway was obtained in 47 patients at the first attempt, but perfect positioning, as assessed by fibreoptic laryngoscopy, was found in only 22 instances. Despite an airway initially patent, delayed airway obstruction occurred in 12 infants. It is concluded that clinical airway patency does not guarantee ideal positioning of LMA in infants, and that care should be taken to ensure continued airway patency because of the tendency of the LMA position to deteriorate in this group of children.

The laryngeal mask airway in children. A fibreoptic assessment of positioning.

Clinical and fibreoptic assessment of the positioning of the laryngeal mask airway was performed in 100 children. Clinical observation indicated a patent airway in 98% and severe airway obstruction in 2% of cases. Perfect positioning, as judged by fibreoptic laryngoscopy, was found in 49% and the epiglottis was within the mask in 49%. Fibreoptic evidence of partial airway obstruction in 17% was not detected clinically.

Differentiation of PC12 cells with K-ras: comparison with nerve growth factor.

The cell line PC12, derived from a rat pheochromocytoma, has served as a model for studies on the mechanism of action of nerve growth factor, as well as for the exploration of neuronal differentiation in general. When treated with nanomolar concentrations of nerve growth factor, these neoplastic chromaffin-like cells stop dividing and acquire, for all intents and purposes, the phenotype of mature sympathetic neurons. This phenotype is characterized by the extensive outgrowth of electrically excitable neurites, the ability to form functional synapses, and the acquisition of a number of biochemical markers. Treatment of PC12 cells with retroviral vectors encoding the K-ras, the N-ras, or the v-src oncogenes also produces a marked morphological differentiation very similar to that seen upon treatment with nerve growth factor. Treated cells stop dividing and develop an extensive network of neurites. It has recently been shown that PC12 cells differentiated with v-src, while resembling, morphologically, those treated with nerve growth factor, differ substantially in the biochemical characteristics normally associated with nerve growth factor-induced differentiation. Cells infected with K-ras also develop a neurite network similar to that seen after treatment with nerve growth factor. In addition, such cells develop tetanus toxin-binding sites and saxitoxin-binding sites, as do cells treated with nerve growth factor. Decreases in the binding of epidermal growth factor and in the activity of calpain also occur and these, as well, are characteristic of nerve growth factor-treated cells. But the adhesive properties of cells infected with K-ras are different than those of nerve growth factor-treated cells, and the former do not show an increase in the NILE glycoprotein. Finally, K-252a, an inhibitor of the actions of nerve growth factor on PC12 cells, has no effect on the neurite outgrowth produced by infection with K-ras. Thus, many of the key markers of nerve growth factor-induced differentiation of PC12 cells also appear upon differentiation with K-ras, but there are, nevertheless, some crucial differences in the properties of these two sets of cells.

Some neuronal properties of PC12 cells differentiated by the K-ras oncogene.

When infected with a virus containing the Kirsten-ras oncogene, rat phaeochromocytoma or PC12 cells elaborated neurites and ceased mitosis, that is, they underwent neuronal differentiation. Such differentiated cells could be replaced and maintained up to 20 weeks in vitro without the need of an exogenous, continuous supply of nerve growth factor (NGF). The neurites of K-ras infected PC12 cells, filled with microtubules and actin which was concentrated within the growth cones, resembled those of primary neurons in vitro. As in the NGF-primed PC12 cells, two types of secretory vesicles were present in the K-ras-infected PC12 neurites: large (100 nm), dense core granules, and small (45 nm), clear vesicles. Compared to naive PC12 cells, K-ras infected PC12 cells had (a) higher activities of acetylcholinesterase and choline acetyltransferase, two enzymes involved in acetylcholine metabolism; (b) enhanced activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis; (c) a higher, evoked norepinephrine release; and (d) similar levels of sodium-dependent uptake of both choline and norepinephrine. Although the total content of catecholamines in K-ras-differentiated PC12 cells was less than that of naïve cells, both norepinephrine and dopamine were present in substantial amounts and norepinephrine was released after stimulation. According to their enzymatic activity, these cells can also synthesize acetylcholine and thus have potential as donors for the intracerebral replacement of either catecholaminergic or cholinergic neurotransmitters.

Intra-oral colonization of macaque monkeys by Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans was acquired by captive Macaca fascicularis 3 to 6 months after birth, and all monkeys aged over 6 months harbored detectable levels. This microorganism was most frequently isolated from the gingival plaque of the incisor (and other) teeth compared with other oral sites. Strains were leukotoxic by bioassay and Western blot analysis. Antibodies in macaque serum contained neutralized the leukotoxin of a human A. actinomycetemcomitans strain. High titres of maternal neutralizing anti-leukotoxin antibodies were detected in neonates; the titre then fell rapidly so that by 6 months the antibody titer was zero. Antileukotoxin antibody production was detected after 6 months of age, rapidly reaching a high level within 2 years after birth. The presence of leukotoxic strains of A. actinomycetemcomitans in the gingival region did not appear to be correlated with an increase in susceptibility to periodontal disease.

Anaesthetic induction with isoflurane or halothane. Oxygen saturation during induction with isoflurane or halothane in unpremedicated children.

The authors performed a randomised, prospective trial in which one junior anaesthetist administered gaseous induction of anaesthesia to 50 unpremedicated children with either isoflurane or halothane in nitrous oxide and oxygen. Arterial oxygen saturation and the electrocardiogram were monitored and the incidence of complications noted. Desaturation below 85% occurred in six children, but only with isoflurane. The incidences of complications and desaturation events did not alter throughout the 25 isoflurane inductions. Coughing, movement, laryngospasm and sinus tachycardia occurred more frequently with isoflurane. Isoflurane inductions took longer (7.9 as compared with 5.4 minutes, p less than 0.001) and had 4.25 times the number of complications.

Killing of human myelomonocytic leukemia and lymphocytic cell lines by Actinobacillus actinomycetemcomitans leukotoxin.

The purified leukotoxin of Actinobacillus actinomycetemcomitans kills human leukemic cell lines (e.g., HL-60, U937, and KG-1) and human T- and B-cell lines (e.g., JURKAT, MOLT-4, Daudi, and Raji) in a dose- and time-dependent manner. The 50% effective doses for these cell lines are similar to those established for human polymorphonuclear leukocytes and monocytes. In contrast, other human and nonhuman tumor cell lines are not susceptible to the leukotoxin. These human leukemia and lymphoid cell lines will serve as useful model systems with which to study the molecular specificity and mechanism(s) of action of the actinobacillus leukotoxin.

Acute beta-adrenoreceptor blockade and induced hypotension.

Thirty patients scheduled for major middle ear surgery and induced hypotension, with a trimetaphan/sodium nitroprusside infusion, were randomly allocated to receive a single oral dose of beta-adrenoreceptor antagonist 2 hours before surgery. In group 1 ten patients received metoprolol 50 mg, in group 2 ten patients received metoprolol 25 mg and in group 3 ten patients received oxprenolol 20 mg. There were ten controls for each group. The nitroprusside infusion rate was significantly reduced in groups 1 and 3. However, there was an unacceptably high incidence of profound bradycardia after induction of anaesthesia in those patients who had received beta-adrenoreceptor antagonists pre-operatively and this technique is not recommended for routine use.

Killing of cultured hepatocytes by conjugates of asialofetuin and EGF linked to the A chains of ricin or diphtheria toxin.

A disulfide-linked conjugate between asialofetuin (ASF) and the toxic A chain (RTA) of ricin is as potent a toxin for cultured rat hepatocytes as our previously described conjugate between ASF and fragment A of diphtheria toxin (DTA). An RTA conjugate of epidermal growth factor (EGF) was a potent toxin for 3T3 cells. In contrast, EGF-DTA was essentially nontoxic for 3T3 cells. We have now examined the toxicity of EGF-RTA and EGF-DTA on cultured hepatocytes. The EGF-DTA conjugate, nontoxic to 3T3 cells, is also a potent toxin for hepatocytes. We also observed a decrease with time of culture in the sensitivity of hepatocytes to the ASF and EGF conjugates. This decrease is not a result of a decrease in EGF or asialoglycoprotein receptors.

Epidermal growth factor binding and mitogenic activity on purified populations of cells from the central nervous system.

Binding of 125I-epidermal growth factor (EGF) to purified populations of rat astrocytes, oligodendrocytes, and neurons was measured. Astrocytes bound 40,000-100,000 EGF molecules per cell, while oligodendrocytes bound only 6,000-10,000 EGF molecules per cell. In contrast, neurons had little or no capacity to bind 125I-EGF. EGF alone was able to stimulate incorporation of tritiated thymidine fivefold in purified astrocyte cultures incubated in serum-free medium. When EGF was added to the previously described chemically defined medium for astrocytes, incorporation of tritiated thymidine in purified astrocytes was equivalent to that observed in medium containing 10% fetal calf serum. Addition of EGF to this chemically defined medium also doubled the proliferative capability of the medium for cultured astrocytes. EGF was maximally effective in stimulating 3H-thymidine incorporation at concentrations between 1-10 ng/ml.

Toxic ligand conjugates as tools in the study of receptor-ligand interactions.

We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time.

Methylenedioxyamphetamine. Clinical description of overdose, death, and review of pharmacology.

Methylenedioxyamphetamine is a commonly used "street drug", with a reputation of providing a "good trip". The drug is structurally similar to both mescaline and amphetamine. The following symptoms were found in a patient: sympathomimetic effects, coma, seizures, hyperreflexia, and hyperthermia. The patient's condition was initially stabilized and then deteriorated with uncontrollable hyperthermia, hematologic abnormalities, and coma that culminated in death. Since the hyperthermia is based on both central and peripheral mechanisms, phentolamine (Regitine) mesylate may be the preferred drug to be used in the treatment of future cases. The concept that this drug is primarily a hallucinogen with mild toxicity is erroneous.

Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes.

We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialogalactoorsomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated.

Glycoenzymes: an unusual type of glycoprotein structure for a glucoamylase.

Glucoamylase, (1 leads to 4)(1 leads to 6)-alpha-D-glucan glucohydrolase (EC 3.2.1.3), hydrolyzes starch and glycogen completely to D-glucose and is used industrially in the manufacture of D-glucose from starch. The enzyme is elaborated by many types of fungi and occurs in two isoenzymic forms (glucoamylase I and glucoamylase II) in extracts from certain fungi. The isoenzymes from Aspergillus niger are glycoenzymes containing D-mannose, D-glucose, and D-galactose as integral structural components. New data from experiments on reductive alkaline beta-elimination and from methylation analyses show that the carbohydrate chains of glucoamylase I are linked O-glycosidically from D-mannose residues to L-serine or L-threonine residues of the protein moiety. In this enzyme, the carbohydrate residues are present as 20 single D-mannose residues, 11 disaccharides components having the structure 2-O-D-mannopyranosyl-D-mannose, 8 trisaccharides, and 5 tetrasaccharides composed of various combinations of D-mannose, D-glucose, and D-galactose residues joined by (1 leads to 3) and (1 leads to 6) glycosidic linkages. Such an array of carbohydrate chains in a glycoprotein is unusual, and may account for some of the unique properties exhibited by glucoamylase.

Age related alterations in the chromosomal proteins from human diploid fibroblasts.

Histone and non-histone chromosomal proteins from human diploid fibroblasts of different in vitro ages were extracted and subjected to SDS--polycrylamide gel electrophoresis. The proteins were taken from cells maintained in three distinct culture states: preconfluent (log phase of growth), confluent (stationary phase of growth), and arrested (presumptive G0 phase). Age associated alterations in the incorporation of radioactive amino acids were detected in the fractionated proteins in every culture state but were less pronounced during the arrested state. Age related difference detected under growth conditions may reflect variations in the proliferative nature of the populations. Differences seen during the arrested state may be indicative of basic changes in the chromosomal protein complement of different age populations.