Selectivity in mobilisation of stored fatty acids by maturing cod, Gadus morrhua L

Lipids, proteins and carbohydrates diminish in the liver and muscle of cod when (a) food is inadequate or (b) the gonads are maturing. The results reported in this paper appear to show that the mobilisation of the lipids differs according to whether the need is just for energy (simple starvation) or for building up gonads. Two groups of fish from the same catch were starved. In one, the gonads were developing, while in the other the gonads had been surgically removed. Significantly more of the fatty acid C22:6 was mobilised from the liver lipids in the group with developing gonads, this fatty acid being present in the greatest amount in the gonads of either sex. The fatty acid C18:1 is also important in the gonads, and this was also preferentially removed from all the livers of maturing-starving fish when compared with gonadectomised-starving, though here the effect was not statistically significant. Some discrimination therefore appears to be exercised when hepatic lipids are removed for gonad development. No selectivity was observed in the mobilisation of fatty acids from the muscle.     

Staining and Histochemistry of Undecalcified Bone Embedded in a Water-Miscible Plastic

Undecalcified bone fixed in a variety of fixatives and embedded in a new formulation of 2-hydroxy propyl methacrylate at 4 C has been sectioned at 1 to 5 microns. The embedding mixture contains 2-butoxyethanol as plasticizer and triethyleneglycol dimethacrylate as cross-linker. The accelerator was benzoyl peroxide and the catalyst was N,N-dimethylaniline. With proper embedding and care in sectioning it is possible to obtain sections with relatively little bone compression, excellent preservation of cellular elements, and a minimum of wrinkling. A wide variety of stains have been used for these sections and those reported here are Gill's hematoxylin-eosin, Nocht's azure-eosin, Feulgen, Hoechst 33258 (bisbenzimid H 33258), methyl green-pyronin, PAS, alizarin red, and von Kossa silver stain. There was excellent preservation of acid and alkaline phosphatase activities. A new method of prestaining immunofluorescent labeling was also applied to bone and examples of staining with anticollagen I and antifibronectin are presented.     

Study of Cholinesterase Active Site Using a Fluorescent Probe

Fluorescence of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride (tacrine) in the presence of butyrylcholinesterase has been studied. Quenching of tacrine fluorescence dependent on the cholinesterase activity has been revealed. A mechanism of quenching is proposed, which involves the formation of a charge-transfer complex including an excited tacrine molecule and indole of the tryptophan residue from the periphery of cholinesterase active site.     

Age-Related Changes in Tyrosine Hydroxylase and Choline Acetyltransferase in Sympathetic Ganglia of a Rat Strain with Reduced Sympathetic Neuron Numbers

Abstract: In Wistar rats, a subpopulation of sympathetic ganglionic neurons dies during ageing, but in the GH strain, these same neurons die during the period of perinatal maturation. We have compared tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in superior cervical ganglia of GH and control rats at different ages. Ganglionic TH rose to near adult levels between postnatal weeks 1 and 2. No significant differences in TH values were seen between GH and control ganglia at any age, indicating that reduced neuron numbers are compensated for by increased cellular activity, Ganglionic ChAT rose initially in parallel with TH and then more slowly over postnatal weeks 3–4, reaching adult levels that were about 20° lower in GH than in normal ganglia. During ageing, TH remained constant but ChAT continued to rise slowly in GH ganglia, whereas ChAT in normal ganglia fell by about 10°. Both the strain difference in ChAT during development and the fall in ChAT during ageing in normal animals parallel the differences in ganglion cell numbers seen under these circumstances.     

Precursors and Metabolites of Norepinephrine in Sympathetic Ganglia of the Dog

3,4-Dihydroxyphenylalanine, dopamine, epinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxyphenylacetic acid as well as norepinephrine were measured in dog lumbar sympathetic ganglia. The responses of these compounds to several classes of stimuli were investigated using an isolated time-resolved superfusion system. Nonselective (i.e., amphetamine and high K+) and receptor-mediated selective (oxotremorine) stimuli were used to evoke releases. The overflows of all compounds were measured by HPLC with electrochemical detection. The efficiency of each stimulus was estimated by normalizing the amount of evoked release to the total neurotransmitter pool when the stimulus was applied; i.e., fractional release was calculated. Overflows of all compounds except 3,4-dihydroxyphenylalanine were enhanced by a 10-min 100 microM amphetamine stimulus, and each of the catecholamine pools (dopamine, norepinephrine, and epinephrine) was affected to the same degree. By contrast, the 3,4-dihydroxyphenylalanine and dopamine pools were more readily releasable than the norepinephrine pool with a 10-min 80 mM K+ stimulus, and these releases were Ca2+ dependent. Epinephrine was released in preference to norepinephrine by a 10-min 1 mM oxotremorine stimulus. The data suggest the existence of at least three types of neurons in dog lumbar ganglia and are consistent with previous histological observations.     

Frequency and Abundance of Alphaherpesvirus DNA in Human Thoracic Sympathetic Ganglia

Alphaherpesvirus reactivation from thoracic sympathetic ganglia (TSG) and transaxonal spread to target organs cause human visceral disease. Yet alphaherpesvirus latency in TSG has not been well characterized. In this study, quantitative PCR detected varicella-zoster virus (VZV), herpes simplex virus 1 (HSV-1), and HSV-2 DNA in 117 fresh TSG obtained postmortem from 15 subjects. VZV DNA was found in 76 (65%) ganglia from all subjects, HSV-1 DNA was found in 5 (4%) ganglia from 3 subjects, and no HSV-2 was found.     

Alanine Uptake and Release by Sympathetic Ganglia of Chicken Embryos

Uptake and release of alanine were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14-15 days old, and incubated in a modified Eagle's minimum essential medium. In the presence of [U-14C]glucose, glucose carbon accumulated in alanine in the medium at a rate that increased when unlabeled alanine was added and sometimes exceeded the rate of appearance in lactate. When combined with uptake data, the increase in appearance of labeled alanine in the medium could be accounted for quantitatively by interference with its reuptake, without assuming a change in the unidirectional output of labeled alanine, provided allowance was made for the measured properties of exchange between the extracellular space and the surrounding medium. According to this model, the constant unidirectional outflux of labeled alanine was about 50 mumol/g dry weight/h. When [U-14C]alanine was added to medium containing unlabeled glucose, the alanine was consumed at a rate that increased as the concentration of alanine in the medium was elevated. The uptake rate was found to fit a modified Michaelis-Menten equation with a Umax of about 120 mumol/g dry weight/h, a Km of 0.5-1.0 mM, and a Kd of 0.75 ml/g dry weight/h. By chemical measurement of changes in alanine concentration in the medium during incubation, the uptake rate was shown to equal the output rate when about 0.2 mM alanine was present. Much of the alanine consumed in the presence of glucose was metabolized to CO2, raising the total CO2 output above the rate obtained with glucose alone. When alanine was present at a concentration of 10-20 mM, it contributed almost as much carbon to CO2 as did the glucose. A higher percentage of the carbon from alanine was incorporated into tissue constituents than was carbon from either glucose or lactate. It is concluded that alanine can be significant both as a product and as a substrate, but that its role as substrate would not be great at typical concentrations of alanine in blood.     

Standardization of Staining in Giycosaminoglycan Histochemistry: Alcian Blue, its Analogues, and Diamine Methods

Glycosaminoglycans are identified in tissue sections by various histochemical techniques including staining with alcian blue and its analogues, such as cuprolinic blue and cupromeronic blue, or with high and low iron diamine methods. The variation in staining results is particularly confusing in the case of alcian blue, where not only are several different brands of alcian blue available but also several different staining protocols are used. If the results obtained by these techniques are compared, they often do not match. We have developed a dot blot technique for quality control of glycosaminoglycan histochemistry to standardize the staining protocols. This staining technique enables his-tochemists to test particular batches of alcian blue or its analogues for selective glycosaminoglycan staining, thus improving control of histochemical results. The results obtained using the dot blot assay indicate that it is necessary to test each batch of dye individually to obtain valid results in glycosaminoglycan histochemistry.     

Fine Structure and Histochemistry of the Choroid Plexus of the Teleost Leuciscus rutilus

Analyses of the fine structure of the posterior choroid plexus in the teleost Leuciscus rutilus and the determination of the presence and function of the enzymes acid and alkaline phosphatases, ATPase and glucose-6-phosphatase confirm similarities between these epithelial cells and the saccus dorsalis and also with the epithelial cells of the choroid plexus found in mammals. The teleost plexus cells contain coated vesicles which are derived from the plasmalemma as well as from the Golgi complex. Moreover, they contain multivesicular bodies and Iysosomes. These organelles function in the absorption of substances from the cerebrospinal fluid and in the breakdown of these substances within the cells. The investigated enzymes play an important role in the secretion of electrolytes into the cerebrospinal fluid by active membrane transport.     

Ontogeny of Glucose and Lipid Metabolism in Dorsal Root Ganglia of Chickens.: Similarities and Contrasts with Sympathetic Ganglia

Dorsal root ganglia, excised from the lumbar roots of the sciatic nerve of white Leghorn chicken embryos 6-13 days of age, were incubated usually for 5 h, at 36 degrees C in 20 microliters of a bicarbonate-buffered physiological salt solution containing 5.5 mM glucose. [U-14C]Glucose, [1-14C]glucose, [6-14C]glucose, or [5-3H]uridine was also added. Lipid synthesis and lactate output were measured by incorporation of 3H from [5-3H]uridine. Glucose uptake and labeled lactate output declined rapidly from 6 to 8-9 days of age, more slowly thereafter. Synthesis of lipids was relatively constant throughout the ages studied, without the increased rate at intermediate ages seen previously in sympathetic ganglia of the same species. RNA synthesis declined progressively throughout the ages studied. The output of C-6 of glucose to CO2 was about the same at all ages, whereas that of C-1 declined rapidly from 6 to 7 days of age and then more slowly, but always remained higher than that of C-6 and thus indicated that much glucose was metabolized via the hexosemonophosphate shunt.     

Fluorescent Staining of Juxtaglomerular Cells in Frozen Sections

Enzymatic investigations of the juxtaglomerular apparatus often creates the need for visualisation of granulated juxtaglomerular cells (JGC) in preparations subjected to histochemical procedures. In our investigations, Pitcock and Hartroft's (1958) modification of Bowie's method and the Endes et al. (1969) combined trichrome staining proved to be inadequate when applied to fresh cryostat sections, or to formol- or glutaraldehyde-fixetl, gum sucrose-impregnated frozen sections. Friedberg and Reid's (1966) crystal violet procedure for waxembedded kidneys also failed to give uniformly reproducible results. In attempting to find a satisfactory technique for both enzyme and granule staining, we noted Janigan's (1965) and Haratla's (1969) observations on paraffin-embedded JGC, and tested the following fluorochromes: thioflavine T—Fluka, C. I. 49005; auramine O—Merck, C. I. 41000; acridine orange—E. Gurr, C. I. 46005; berberine sulfate—Fluka, C. I. 75160 on 10 μ sections of albino mouse kidneys prepared in 4 different ways as follows:     

Sympathetic nervous control of the iris sphincter of the atlantic cod,Gadus morhua

Summary The autonomic nervous control of the cod iris has been studied. The pharmacological properties of the smooth muscles of the iris have been elucidated by agonist/antagonist studies on isolated strip preparations. Electrical stimulation of parasympathetic and sympathetic pathways to the eye have been carried out, with recordings of the movements of the iris margin. Additions of cholinergic and adrenergic antagonists in selective concentrations were made to investigate the nature of the autonomic nerve fibres controlling the iris.Isolated strip preparations of the iris sphincter contracted in response to cholinergic or-adrenoceptor agonists. There appear to be no radial muscular elements in the cod iris. The effect of carbachol on the iris sphincter could be competitively antagonized by atropine, suggesting the presence of muscarinic receptors of the smooth muscles. The effect of adrenaline was similarly antagonized by phentolamine. The effect of phentolamine, and the order of potency for the adrenergic agonists, shows the presence of-adrenoceptors in the iris sphincter.-adrenoceptors of minor importance are also suggested by the inhibitory effects of isoprenaline on preparations pre-contracted by carbachol.The indirectly acting adrenergic agonist tyramine also contracts the isolated sphincter preparations. This effect is probably due to release of nervously stored catecholamines, since tyramine lacks effect on preparations from animals pre-treated with 6-hydroxydopamine. Preparations from 6-hydroxydopamine pre-treated animals also show a 10-fold increase in the affinity for adrenaline, demonstrating the development of a pre-synaptic supersensitivity due to the destruction of adrenergic nerve terminals of the iris. Stimulation of the sympathetic chain or ciliary nerves produces a constriction of the pupil of the same side. Application of selective concentrations of the antagonists atropine and phentolamine shows that the sympathetic constrictory innervation is solely adrenergic. In some preparations a small pupillo-dilatory effect of nerve stimulation is evident after the constrictory effect has been abolished by phentolamine. This inhibitory effect can be abolished by propranolol, indicating the presence of a-adrenoceptor mediated inhibitory control of minor importance. Stimulation of the oculomotor nerve produces no consistent responses of the cod iris.Illumination of one eye produces a pupilloconstriction comparable to that seen after sympathetic nerve stimulation. The light induced response is insensitive to atropine, phentolamine and tetrodotoxin, showing a direct effect on the smooth muscles of the sphincter. There is no consensual reflex in the cod.I wish to thank Dr. Susanne Holmgren for critically examining the original draft of this paper, and Mrs. Lena Utter for skilled assistance with isolated strip preparations and processing of concentration-response data. The fish was kindly supplied by Mr. Ingmar Hakemar. This work has been supported by grants from the Swedish Natural Science Research Council, the M. Bergvall Foundation and the Adlerbert Foundation.     

Evaluation of a Fluorescent Lectin-Based Staining Technique for Some Acidophilic Mining Bacteria

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245–2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.     

Development of a Vital Fluorescent Staining Method for Monitoring Bacterial Transport in Subsurface Environments

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10⁵ CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.     

Lactate Uptake and Release in the Presence of Glucose by Sympathetic Ganglia of Chicken Embryos and by Neuronal and Nonneuronal Cultures Prepared from These Ganglia

Abstract: Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6–17 mM glucose, various concentrations of lactate, and either [U-¹⁴C]lactate, [1-¹⁴C]glucose, or [6-¹⁴C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis-Menten equation, with V_max = 360 μmol/g protein/h and K_m = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO₂. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady-state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO₂ and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood-brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.