A critical evaluation of the use of filipin-permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al. retained 65-75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen. We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin-treated cells. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.     

Time Course of Adaptations in Dopamine Biosynthesis, Metabolism, and Release Following Nigrostriatal Lesions: Implications for Behavioral Recovery from Brain Injury

Alterations in neostriatal dopamine metabolism, release, and biosynthesis were determined 3, 5, or 18 days following partial, unilateral destruction of the rat nigrostriatal dopamine projection. Concentrations of dopamine and each of its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) were markedly decreased in the lesioned striata at 3, 5, or 18 days postoperation. The decline in striatal high-affinity [3H]dopamine uptake closely matched the depletion of dopamine at 3 and 18 days postoperation. However, neither DOPAC, HVA, nor 3-MT concentrations were decreased to as great an extent as dopamine at any time following lesions that depleted the dopamine innervation of the striatum by greater than 80%. In these more severely lesioned animals, dopamine metabolism, estimated from the ratio of DOPAC or HVA to dopamine, was increased two- to four-fold in the injured hemisphere compared with the intact hemisphere. Dopamine release, estimated by the ratio of 3-MT to dopamine, was more increased, by five- to sixfold. Importantly, the HVA/dopamine, DOPAC/dopamine, and 3-MT/dopamine ratios did not differ between 3 and 18 days postlesioning. The rate of in vivo dopamine biosynthesis, as estimated by striatal DOPA accumulation following 3,4-dihydroxyphenylalanine (DOPA) decarboxylase inhibition with NSD 1015, was increased by 2.6- to 2.7-fold in the surviving dopamine terminals but again equally at 3 and 18 days postoperation. Thus, maximal increases in dopamine metabolism, release, and biosynthesis occur rapidly within neostriatal terminals that survive a lesion. This mobilization of dopaminergic function could contribute to the recovery from the behavioral deficits of partial denervation by increasing the availability of dopamine to neostriatal dopamine receptors. However, these presynaptic compensations are not sufficient to account for the protracted (at least 3-week) time course of sensorimotor recovery that has been observed following partial nigrostriatal lesion.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Phenobarbital and 6-aminonicotinamide effect on cerebral enzymatic activities related to energy metabolism in different rat brain areas

The effect of phenobarbital (100 mg/kg i.p.) and 6-aminonicotinamide (6AN) (35 mg/kg i.p.) on enzyme activities related to energy transduction was investigated on the homogenate in toto, non-synaptic mitochondrial fraction and synaptosomal fraction isolated from different rat brain areas (cerebral cortex, hippocampus, hypothalamus, striatum, and medulla oblongata). 6AN treatment decreased: (a) phosphofructokinase in all the areas tested; (b) lactate dehydrogenase on the homogenate in toto in striatum and hypothalamus, and on the synaptosomal fraction in cerebral cortex and corpus striatum; (c) succinate dehydrogenase on non-synaptic mitochondrial fraction in hippocampus and striatum. Finally, aspartate aminotransferase was increased on non-synaptic mitochondrial fraction in striatum and medulla oblongata. Phenobarbital treatment induced an increase of total NADH cytochrome c reductase on mitochondrial fraction in hippocampus and hypothalamus, and a decrease of cytochrome oxidase activity on non-synaptic mitochondrial fraction in hypothalamus and medulla oblongata.     

冷冻或切除小脑蚓部山顶(Culmen)对豚鼠“踏步自动作用”(Stepping automatism)的影响

本研究探讨部分冷冻或切除小脑蚓部(vermis)对整体豚鼠“踏步自动作用”(steppingautomatism)的影响。“踏步自动作用”由我们近年来发现的诱发踏步物质(SIS)(4-R-2,2,5,5-四(三氟甲基)-咪唑啉)所引起。结果表明部分冷冻或切除小脑蚓部的山顶(culmen,Ⅴ和Ⅳ叶)和中央叶(Centralis,Ⅲa,b)明显增强豚鼠的“踏步自动作用”。冷冻小脑不能触发,但仅能调控“踏步自动作用”。这种调控作用对自动化程度差的弱“踏步自动作用”特别显著。蚓部山顶(Ⅴ叶为主)同时调控左右前肢踏步,而一侧蚓部山顶及其半球则主要调控同侧前肢踏步。此外,本研究的结果表明当介面温度(冷冻头和小脑幕间)致冷至5℃—0℃左右,冷冻小脑便可基本模拟部分切除小脑效应。     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline"

Summary Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly (mutation frequency decline or MFD), whereby post-irradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 by region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 by sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6–4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6–4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6–4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.