摘要:

A novel and rapid procedure is described for the preparation of chromatin‐depleted nuclei (CDN) from Friend erythroleukemia cells under conditions that avoid the use of high salt concentrations. By this procedure isolated nuclei that had previously been incubated with DNase I to partially digest DNA were washed twice in 2 mM EDTA to extract the chromatin. The resulting structures contained 1% of DNA, 65% of total RNA, 60‐80% of hnRNA, 74% of snRNA, 29% of protein, and 2% of histones contained in isolated nuclei. Electron microscopy revealed intact, spherical structures similar in diameter to isolated nuclei and consisting of dense networks of fibrils 50‐100 A thick surrounded by distinct nuclear laminae. Although no morphological evidence was found for residual nucleoli, C23, a nucleolus‐specific phospho‐protein, remained centrally localized in CDN, while Sm antigen specific for snRNPs was diffusely localized but absent from central regions. Addition of 2 mM MgCl2 to CDN resulted in the reformation of morphologically distinguishable residual nucleoli. These studies suggest that nucleolar morphology is, in part, dependent upon divalent cations and components unique to the nucleolar matrix and demonstrate that little randomization of nuclear and nucleolar matrix fibrils occurs during CDN isolation in the absence of divalen

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00204.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

Chromatin‐depleted nuclei (CDN) were prepared from Friend erythroleukemia cell nuclei by partial digestion with DNase I and extraction of the chromatin by 2 mM EDTA as described in the preceding paper (Long and Ochs, 1983. Biol. Cell 48, 99‐108). These structures contained dense networks of matrix fibrils surrounded by distinct laminae but no morphologically distinct residual nucleoli. CDN disrupted by gentle shearing or 1 microgram/ml RNase were fractionated into laminae and matrix fibrils by differential centrifugation. Protein composition of the lamina fraction was dominated by two prominent lamina proteins that were not detectable in the matrix fraction. Mild RNase treatment led to a conversion of the fibrous network to a particulate morphology while mild shearing resulted in an apparently unaltered fibril fraction. The matrix fibril fractions contained hnRNP proteins and the snRNAs. These results suggest that EDTA‐prepared CDN may provide a system for studying snRNP‐hnRNP interactions and hnRNP processing that is less complex than intact

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00205.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

A method is described for preparation of a fraction of chromatin enriched in transcribing regions from nuclei of mouse GR cells. This fraction, released by mild staphylococcal nuclease digestion of isolated nuclei, contains 2 to 10% of the DNA as polynucleosomal chromatin together with 50‐70% of pulse‐labelled RNA and about 90% of all template‐engaged RNA polymerase B molecules, titrated with (3H)‐alpha‐amanitin. Hybridisation of DNA from this chromatin fraction to total nuclear RNA in excess shows that it is enriched in frequently‐transcribed DNA sequences. A modification of the Miller technique, allowing the spreading of the active chromatin fraction for electron microscopy, has been developed. Examination of the spreads reveals that this chromatin fraction contains 20‐100 nucleosome‐long polynucleosomal chains bearing lateral RNP fibrils interpreted as nascent transcripts. The average length of the DNA fragments in the fraction is greater than that of average transcribed regions, suggesting that the transcribed regions are linked to flanking segments whose chromatin conformation probably contributes to the selective release of transcr

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00206.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

L Pyruvate kinase (LPK) is considered to be the major form in the liver. Two isozymes, LPK and MPK, have been localized in the isolated rat hepatocyte in vitro with an immunocytometric method. MPK is induced by insulin, which also creates a slight stimulation of LPK (at physiological doses) in both fed and fasted animals. Glucagon inhibits LPK in fed animals (the fasting rat is already in a situation of gluconeogenesis and this hormone is ineffective). MPK is insensitive to glucagon, regardless of the nutritional state of the animals. Each PK isozyme is thus controlled predominantly by one of the two hormones, corresponding to a sophisticated regulation of hepatic glycolysis and gluconeogenesis.

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00207.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

The uptake of a fluorescent phallotoxin (tetramethylrhodaminyl‐phalloidin) into rat hepatocytes has been studied. The experiments were performed in vitro, using freshly isolated hepatocyte suspensions or monolayers of hepatocytes cultured for up to 5 days, as well as in vivo, by investigating cryostat sections of a liver from an animal injected with the labelled toxin. In vitro, in freshly isolated hepatocytes, a staining of actin was observed. On the contrary, if the hepatocytes were cultured, only fluorescent endocytotic vesicles were found accumulated around the nucleus, and remaining in the cells unchanged for several days. In vivo, both fluorescent patterns were observed, often in one and the same cell. The endocytotic vesicles of rhodaminylphalloidin looked very similar to those obtained with fluoresceinyl‐concanavalin A. navalin A. We conclude that in all systems the fluorescent phallotoxin enters the hepatocytes by endocytosis. However, in the freshly isolated cells the endocytotic vesicles apparently undergo some kind of processing with release of the toxin and subsequent staining of cellular actin, while in cultured hepatocytes the endocytotic vesicles persist unproces

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00208.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

Most of mouse diaphragm muscle acetylcholinesterase (AChE) is irreversibly inhibited after a single intraperitoneal injection of a methyl‐phosphorothiolate derivative (MPT), an organophosphorus compound which phosphorylates the active site. The muscle recovers its AChE (de novo synthesis) and we studied the time course of reappearance of AChE and its multiple active molecular forms. After inhibition, there is an initial (3 to 15 hr) rapid recovery of total AChE (which evolves from 20‐28% to 50‐60% of the control values), followed by a slow phase of AChE return. After 3 days, the recovery is still incomplete (reaching 70‐80% of control values). Among the main molecular forms present in diaphragm muscle (16 S, 10 S and 4 S, accompanied by minor components), the 16 S and 10 S forms are the most sensitive to MPT treatment. During the rapid initial phase of AChE recovery, the absolute rate of recovery of the 4 S form is faster than for the other forms with a correspondingly much higher relative proportion to total AChE. These observations are consistent with the hypothesized precursor role of the 4 S form. The 16 S form, which is found concentrated in the motor end‐plate (MEP)‐rich regions and in low amounts in MEP‐free regions, is similarly partially recovered in both regions, suggesting that there is 16 S biosynthesis not only in the MEP‐rich regions but also in the M

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00209.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

The variations of several biogenic amines in brown adipose tissue (BAT) during cold exposure were studied and their localization investigated with histological methods. The study of serotonin and its metabolite 5‐HIAA suggests that BAT serotonin is mobilized during acute and chronic cold exposure. This amine was found to be principally stored, together with histamine, in mast cells. The mast cell number in BAT was doubled during cold adaptation, as was the histamine content of the tissue. Using radio‐enzymatic assay and high pressure liquid chromatography, only small amounts of dopamine were found in BAT. Since no specific dopamine‐storing structure was detected (for example SIF cells), this low amount of dopamine is probably the precursor pool for noradrenaline synthesis and is most likely stored in the noradrenergic innervation of the tissue. BAT is known to be sensitive to both exogenous serotonin and exogenous dopamine; according to our results serotonin could play a role in BAT regulation while the role of dopamine remains hypothe

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00210.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

The relationship between brown adipose tissue (BAT) and its sympathetic innervation during development was investigated by transplantation of undifferentiated (white fat‐like) hamster BAT into the anterior eye chamber of adult hamsters. Such transplants are known to be revascularized and reinnervated by the vessels and the nerves of the host iris. The morphology of the BAT transplants was analysed during the post‐operative weeks by light and electron microscopy, and the ingrowth of sympathetic nerve fibres from the iris was followed by radioautography. BAT appeared to differentiate in oculo, i.e. presented increasing amounts of adipocytes with multilocular fat deposits and abundant, well‐developed mitochondria, but only after a delay of approx. 10 days, and remained much fatter than in situ. The establishment of the sympathetic innervation was not synchronous with the revascularization process. It occurred simultaneously with the morphological differentiation of the BAT transplants, and the nerve fibre density remained low. In the absence of sympathetic innervation, i.e. when the host irides were sympathectomized prior to transplantation, BAT still differentiated, but the process was further delayed and the proportion of differentiated brown adipocytes after 20 days in oculo was clearly lower than in control transplants. It is concluded that the sympathetic innervation in BAT is involved in the regulation of differentiating activity in the tissue, but is not obligatory for differentiation to

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00211.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

The outermost Golgi cisterna (OGC) frequently exhibits 55 nm diameter protuberances in P fracture faces and corresponding depressions, or pits, in E faces. When these protuberances (pits) appear regularly disposed, OGC faces are recognizable at relatively low electron microscopic magnifications. The mean particle width is less in OGC (6.4 nm) than in rough ER (7.9 nm) P faces, while particle number per unit area is respectively 70% and 100% greater in OGC P and E faces than in corresponding ER faces. The rather small OGC P face particles are better resolved at a relatively low shadowing angle. These differences in particle size (for P faces) and density between OGC and rough ER faces are detectable by inspection and may be used in the recognition of OGC faces devoid of protuberances (in P faces) and pits (in E faces). Fusion of presumably rough ER‐derived microvesicles form short double‐bossed tubular elements which constitute the OGC initial structure. These tubules enlarge by addition of microvesicle membrane and contents forming a characteristic sheet‐like bossed stru

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00212.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY

摘要:

The albumin, orosomucoid and alpha 2‐macroglobulin secretion by isolated hepatocytes of normal and suffering from Turpentine‐induced inflammation rats, is investigated for 4 hr. The model, stable over the whole duration of incubation, is a true reflect of hepatic secretion in vivo and can be used to measure

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1984.tb00213.x

出版商:Blackwell Publishing Ltd    

年代:1984

数据来源: WILEY