PROTEOLYTIC ACTIVITY AND BASIC PROTEIN LOSS IN AND AROUND MULTIPLE SCLEROSIS PLAQUES: COMBINED BIOCHEMICAL AND EIISTOCHEMICAL OBSERVATIONS

Abstract— This combined histochemical and biochemical study has shown that acid proteinase activity (PH 3.5) is increased around histologically-defined active plaques of multiple sclerosis (MS). Biochemical estimation showed that the enzyme is more active in most samples of 'normal' white matter in MS than in controls. A gradient of enzyme activity was observed: control white matter-white matter distant from plaqueclose white matter-edgsplaque. Both electrophoretic and histochemical techniques revealed a reduction or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base-line of thin-layer chromatoplates were shown to be predominantly phosphoinositides combined with encephalitogenic protein     

HISTOCHEMISTRY OF MYELIN—XII ANIONIC STAINING OF MYELIN BASIC PROTEINS FOR HISTOLOGY, ELECTROPHORESIS AND ELECTRON MICROSCOPY

Abstract— Phosphotungstic acid haematoxylin, trypan blue and amidoblack techniques have been developed as anionic dye methods for staining myelin basic proteins. All methods displayed central and peripheral nervous system myelin in histochemical prepa rations and stained brain basic proteins in electrophoretic polyacrylamide gels: phosphotungstic acid haematoxylin appeared to be the most selective of these techniques. Electron photomicrographs of peripheral nerve stained by phosphotungstic acid haematoxylin showed that the major part of myelin basic protein is located in the period dense line. The basic proteins stained by phosphotungstic acid haematoxylin showed an early loss in rat sciatic nerve undergoing Wallerian degeneration and had completely disappeared from the centre of 20 plaques of multiple sclerosis.     

The Immunological Identification of Brain Proteins on Cellulose Nitrate in Human Demyelinating Disease

Abstract: An immunological technique has been employed to identify proteins, separated in polyacrylamide gels, which show changes in brain samples from cases of multiple sclerosis and subacute sclerosing panencephalitis. Sodium dodecylsulphate-treated proteins in particulate and soluble fractions were separated in polyacrylamide slab gels, transferred electrophoretically onto cellulose nitrate sheets, incubated with specific antisera and visualized by an immunoperoxidase method. Protein bands showing changes were identified using antisera raised against the myelin basic and Wolfgram proteins, the neurofilament triplet proteins, tubulin and glial fibrillary acidic protein. In addition to the loss of myelin proteins, decreases in the neurofilament proteins and in tubulin were seen in both multiple sclerosis and subacute sclerosing panencephalitis samples. The distribution of glial fibrillary acidic protein polypeptides in the particulate and soluble fractions of plaque samples appeared to vary according to the degree of fibrosis. Changes in the levels of the myelin-associated glycoprotein, the lower molecular weight component of the Wolfgram protein, albumin and immunoglobulin G, none of which were visualized by protein staining, were also seen. This immunological technique has allowed a closer examination of changes occurring in brain protein spectra in multiple sclerosis and subacute sclerosing panencephalitis.     

The quantitative histochemistry of multiple sclerosis plaques: acid proteinase and other acid hydrolases.

Abstract— Sensitive micromethods were used to study the plaques, adjacent white matter and remote, grossly normal white matter from two cases of multiple sclerosis and to compare them with white matter from normal controls. Lipid-free dry wt/unit of volume was found to be similar for plaques and for normal white matter, reflecting a high water content of plaque tissue and establishing a base for comparison of enzyme activities. Elevations of acid proteinase in and around plaques were confirmed, but they were far exceeded by the increases in acid phosphatase; other acid hydrolases (β-galactosidase, β-glucuronidase and dipeptidyl arylamidase II) showed no significant or consistent changes. However, an acid lipase-esterase hydrolysing 4-methylumbelliferyl oleate was about 30% as active in plaques as in normal-appearing white matter. Glucose-6-phosphate dehydrogenase was unchanged except in one plaque, but lactic dehydrogenase was markedly elevated both in plaques and adjacent white matter. The grossly normal white matter of MS patients, although histologically far from normal (showing gliosis, perivascular infiltrations and small plaques), did not differ significantly from controls with regard to the activity of any of the enzymes studied. DNA levels were much reduced in plaques, but comparisons were difficult because of the apparent gliosis in normal white matter. Decreases in dry wt/unit vol, reflecting partial demyelination, could be shown to extend in a gradient to a distance of about 2 mm. from the edge of certain plaques.     

Proteolysis and myelin breakdown: a review of recent histochemical and biochemical studies

Synopsis Proteins are important constituents of the myelin sheath and serve to maintain its structural integrity. One of the protein components is susceptible to tryptic digestion and may be regarded as a particularly vulnerable part of the myelin sheath. The initial events in myelin breakdown may involve disruption of lipid-protein attachments followed later by chemical degradation of released lipids.In Wallerian degeneration the activity of proteolytic enzymes increases by 12 hr after nerve section. Proteolytic enzyme activity increases in the prodromal phase of diphtheritic neuropathy. Extracts of degenerating nerve cause proteolysis of normal myelin with loss of trypanophilic basic protein and lipid; selective loss of basic protein occurs very early in Wallerian degeneration and has also been found in and around plaques of multiple sclerosis. Proteolytic activity is increased at the edges of active multiple sclerosis lesions. It has been shown that the basic encephalitogenic protein is susceptible to digestion by neural proteases, yielding an active encephalitogenic fragment.It is inferred from these collective observations that proteases play an important role in early myelin breakdown and may also be implicated in the pathogenesis of multiple sclerosis plaques by digesting basic protein, by releasing lipid from its attachment to such protein, and by liberating an active encephalitogenic peptide. The factors responsible for the activation and release of proteases remain unknown.Research Associate supported by the Multiple Sclerosis Society.     

Histochemistry of myelin. XI. Loss of basic protein in early myelin breakdown and multiple sclerosis plaques

Synopsis the early stages of Wallerian degeneration in peripheral nerves are accompanied by loss of a trypanophilic, trypsin-digestible basic protein from myelin. This loss of basic protein is ascribed to the activity of proteolytic enzymes. The reduced trypanophilia in degenerating nerves could not be attributed to loss of lipid. Likewise, the tryptophan-rich trypsin-resistant neurokeratin component of peripheral nerve myelin showed no change in the first week of degeneration. Loss of basic protein has been observed in and surrounding plaques of multiple sclerosis. We infer that digestion of basic protein would lead to the release of the encephalitogenic antigen contained therein.Research Associate supported by the British Multiple Sclerosis Society.     

High Resolution Proton NMR Spectroscopy of Multiple Sclerosis Lesions

Abstract: Tissue from postmortem multiple sclerosis and normal control brains was extracted with perchloric acid and analysed using proton NMR spectroscopy. The content of N -acetyl-derived groups (the sum of N -acetylaspartate, acetate, and N -acetylaspartylglutamate) was decreased in multiple sclerosis plaques compared with normal control white matter (mean, 4.36 vs. 6.64 µmol/g wet weight). In normal appearing white matter adjacent to plaques a corresponding decrease was seen, with no change in white matter distant from plaques. A decrease in the content of total creatine was observed in multiple sclerosis plaques in comparison with normal control white matter (mean, 4.64 vs. 6.56 µmol/g wet weight), which correlated strongly with the decrease in N -acetyl-derived groups. No changes in other metabolites such as total choline or myo -inositol were seen. The decreases in content of N -acetyl-derived groups are in agreement with observations from in vivo proton NMR spectroscopy in multiple sclerosis patients. The decrease in total creatine is in contrast to most of the observations made in vivo where total creatine is assumed to be unchanged and metabolite levels are often expressed as a total creatine ratio. The use of a total creatine ratio in vivo could lead to an underestimation of reductions in N -acetylaspartate and an apparent increase in other metabolites in the multiple sclerosis lesion.     

White Matter Proteins in Multiple Sclerosis

Abstract: The SDS-soluble membrane proteins of plaques and of macroscopically normal white matter from multiple sclerosis brain were investigated by gradient polyacrylamide gel electrophoresis (PAGE). Eleven protein bands were analyzed in detail. The extensive loss of myelin proteins in plaque samples was accompanied by changes in at least three other non-myelin proteins, besides glial fibrillary acidic protein (GFAP), which probably reflect gliosis. Densitometric analysis of the PAGE patterns of membrane fractions from MS and control white matter revealed significant quantitative differences in a number of protein bands. A reduction in myelin basic protein (BP) was associated with an equally significant increase in a high-molecular-weight peptide fragment which may prove to be a breakdown product of BP. Small but highly significant differences in the Wolfgram protein and in one non-myelin protein were also a consistent feature of the normal-appearing white matter samples. The problem of defining normal white matter in multiple sclerosis brain is discussed in relation to the results of the present study, which suggest that one of the early events in the pathogenesis of the disease prior to frank demyelination is an alteration in the protein components of the myelin sheath and possibly of glial cells.     

GANGLIOSIDE ABNORMALITIES IN MULTIPLE SCLEROSIS

Abstract— Gangliosides were isolated from plaque tissue and normal appearing white matter of multiple sclerosis (MS) brain. All four plaques showed decreased ganglioside concn relative to normal human white matter on a wet wt basis, but significant elevation in terms of dry wt. The wet wt and dry wt concn of MS white matter gangliosides showed smaller but statistically significant decreases below normal. Thin-layer patterns of the plaques showed several departures from normal white matter, including decrease of G₄ and G₅, and complete loss of G₇ (sialosylgalactosylceramide). Most of the plaques had significant elevation of G_2A and G_3A along with increases of the slower-migrating polysialogangliosides. An additional ganglioside was present between G₂ and G_2A which was not seen in normal white matter. The TLC pattern of MS white matter gangliosides was essentially normal. The evidence for a general decrease of acidic lipids within normal appearing white matter is discussed.     

Immunoregulatory molecules and IL 2 receptors identified in multiple sclerosis brain

Frozen brain specimens from eight multiple sclerosis (MS) patients were examined for the presence of leukocytes and their cell products by using a panel of monoclonal antibodies. The results presented here demonstrated that in the MS lesion, there was a marked accumulation of HLA-Dr-positive (Ia-positive) cells. These Ia-positive cells were identified as being glial fibrillary acidic protein positive by using double staining methods. Furthermore, the cells in the MS lesion expressed the interleukin 2 (IL 2) receptor, as identified by the anti-TAC monoclonal antibody. The cells in the region of the plaque also exhibited positive staining with antibodies to IL 1, IL 2, and prostaglandin E. Neither normal brain nor brain specimens from progressive multifocal leukoencephalopathy or Alzheimer's disease showed such patterns of staining. These results suggest that stimulated cells are present in the MS brain, thus implicating an active immune mechanism in the pathogenesis of MS.     

HISTOCHEMISTRY OF MYELIN–XV. CHANGES IN THE MYELIN PROTEINS OF THE PERIPHERAL NERVE UNDERGOING WALLERIAN DEGENERATION–ELECTROPHORETIC AND MICRODENSITOMETRIC OBSERVATIONS

The chronological order of changes in rat peripheral nerve proteins during Wallerian degeneration has been investigated by microdensitometric and electrophoretic techniques. Both methods revealed an early loss of myelin proteins. The histochemical microdensitometric study showed a very substantial early loss of stainable protein basic groups and a somewhat slower progressive loss of the major protein component of peripheral nerve myelin (the J band). The electrophoretic study showed an early loss of both the J band protein and the slower-moving basic protein band. The histochemical study also suggested that some cerebroside may be lost in the early stage of Wallerian degeneration. It is concluded that degradation of myelin proteins is an initial event in the process of myelin breakdown.     

Hypertension exacerbates coronary artery disease in transgenic hyperlipidemic Dahl salt-sensitive hypertensive rats

BACKGROUND: The mechanisms underlying the known interaction of two complex polygenic traits, hypertension and hyperlipidemia, resulting in exacerbation of coronary artery disease have not been elucidated. Identification of critical pathways underlying said exacerbation could identify mechanism-based targets for intervention and prevention. MATERIALS AND METHODS: To investigate hypertension- atherosclerosis interaction, we studied the inbred transgenic atherosclerosis-polygenic hypertension Dahl salt-sensitive (S) rat model (Tg53), which over-expresses human cholesteryl ester transfer protein (hCETP) in the liver, and exhibits coronary artery disease and decreased survival compared with control non-transgenic Dahl S rats. Using serial-section histopathological and immunohistochemical analyses, we analyzed the coronary artery disease phenotype of Tg53 rats at end-stage marked by cardio-respiratory compromise as the experimental equivalent of acute coronary syndromes, and determined the effects of reduction of blood pressure through low salt diet (0.008% NaCl) on the coronary artery disease phenotype and survival. RESULTS: End-stage Tg53 rats exhibit coronary artery lesions in the proximal right coronary artery system which exhibit "culprit plaque" features such as plaque inflammation, matrix degradation, apoptosis, neovascularization, thrombosis and hemorrhage recapitulating said features and heterogeneity of human coronary "culprit plaques". Comparative analysis of 6 month vs end-stage lesions reveals distinct lesion development profiles of proximal coronary lesions which quickly progress from eccentric non-occlusive foam-cell rich lesions at 6 months to occlusive "culprit plaques", compared with more distal coronary lesions which exhibit occlusive thick-cap atheroma that remain relatively unchanged from 6 months to end stage. Reduction of hypertension through a low-salt (0.008% NaCl) diet increased survival (P < 0.0001) of Tg53 rats and significantly attenuated the coronary artery disease phenotype detected at 10 months of age marked by diminished apoptosis, neovascularization, matrix degradation compared with end-stage lesions detected at <8 months of age. CONCLUSIONS: End stage coronary lesions in the Tg53 rats recapitulate many, albeit not all, features of "culprit plaques" in humans supporting proposed paradigms of plaque vulnerability implicating lesion macrophage enrichment, apoptosis, matrix degradation and pathological neovascularization. Comparative time course analysis of coronary lesions reveals that plaques which develop into end-stage "culprit plaques" are distinct from "stable plaques" by location and early lesion morphology, suggesting distinct lesion development and progression pathways. The significant effects of low-salt diet-induced decrease in hypertension on right coronary disease phenotype provides compelling evidence that polygenic hypertension accelerates coronary plaque progression and complication independent of cardiac hypertrophy, and more importantly provides paradigmatic support for public health policy.     

Complement factor C5a induces atherosclerotic plaque disruptions

Complement factor C5a and its receptor C5aR are expressed in vulnerable atherosclerotic plaques; however, a causal relation between C5a and plaque rupture has not been established yet. Accelerated atherosclerosis was induced by placing vein grafts in male apoE^?/? mice. After 24 days, when advanced plaques had developed, C5a or PBS was applied locally at the lesion site in a pluronic gel. Three days later mice were killed to examine the acute effect of C5a on late stage atherosclerosis. A significant increase in C5aR in the plaque was detectable in mice treated with C5a. Lesion size and plaque morphology did not differ between treatment groups, but interestingly, local treatment with C5a resulted in a striking increase in the amount of plaque disruptions with concomitant intraplaque haemorrhage. To identify the potential underlying mechanisms, smooth muscle cells and endothelial cells were treated in vitro with C5a. Both cell types revealed a marked increase in apoptosis after stimulation with C5a, which may contribute to lesion instability in vivo. Indeed, apoptosis within the plaque was seen to be significantly increased after C5a treatment. We here demonstrate a causal role for C5a in atherosclerotic plaque disruptions, probably by inducing apoptosis. Therefore, intervention in complement factor C5a signalling may be a promising target in the prevention of acute atherosclerotic complications.     

Gene microarray analysis of multiple sclerosis lesions

Multiple sclerosis (MS) is an autoimmune disease affecting central nervous system white matter. The cause is unknown. It is thought that environmental factors trigger an immune response against myelin antigens in a genetically susceptible individual. The characteristic lesion of MS seen in the brain is a plaque, an area of inflammation, demyelination and glial reaction or ‘sclerosis’. Several recent studies have examined gene expression in MS plaques on a large scale using microarray technology. The involvement of immune-related genes has been confirmed, and many new genes not previously associated with MS lesions have been identified. Microarray studies are significant in identifying potential new targets for therapy.     

Lipid microarrays identify key mediators of autoimmune brain inflammation

Recent studies suggest that increased T-cell and autoantibody reactivity to lipids may be present in the autoimmune demyelinating disease multiple sclerosis. To perform large-scale multiplex analysis of antibody responses to lipids in multiple sclerosis, we developed microarrays composed of lipids present in the myelin sheath, including ganglioside, sulfatide, cerebroside, sphingomyelin and total brain lipid fractions. Lipid-array analysis showed lipid-specific antibodies against sulfatide, sphingomyelin and oxidized lipids in cerebrospinal fluid (CSF) derived from individuals with multiple sclerosis. Sulfatide-specific antibodies were also detected in SJL/J mice with acute experimental autoimmune encephalomyelitis (EAE). Immunization of mice with sulfatide plus myelin peptide resulted in a more severe disease course of EAE, and administration of sulfatide-specific antibody exacerbated EAE. Thus, autoimmune responses to sulfatide and other lipids are present in individuals with multiple sclerosis and in EAE, and may contribute to the pathogenesis of autoimmune demyelination.     

Intravascular ultrasound imaging of myocardial-infarction-related arteries after percutaneous transluminal coronary angioplasty reveals significant plaque burden and compensatory enlargement

We studied patients with acute myocardial infarction (MI) by intravascular ultrasound (IVUS) to elucidate the controversy as to the amount and severity of the atherosclerotic disease at the culprit lesion site in acute MI, as discrepancies exist between angiographic and pathological reports. Twenty-five consecutive patients (age 56 3 10.5 years), with acute MI, underwent IVUS study of the MI-related artery immediately following successful PTCA to the culprit lesion. The IVUS images were analyzed quantitatively and qualitatively and were compared with the angiography of the same arteries. At the PTCA site, 64% of the lesions had an area stenosis of 50-70% and the plaque cross-sectional area (CSA) averaged 0.5 3 0.18 of the arterial CSA. IVUS-defined atherosclerosis was found also in 72% of the segments proximal and distal to the culprit lesion with a plaque/artery CSA ratio of 0.25 3 0.2. The angiogram revealed only 30% of these segments to be abnormal (P 3 0.001). Sixty-nine per cent of all the plaques were defined as 'soft' (low echo-genecity) versus 31% 'hard' (high echo-genecity). The hard plaques were larger than the soft plaques (0.5 3 1.6 versus 0.37 3 0.19 CSA index, respectively, P 3 0.01). With the increase in plaque area there was a significant increase in arterial cross-sectional area. This was demonstrated for all the diseased segments with a correlation coefficient of 0.49 (P 3 0.0001) and for the diseased reference sites a similar correlation coefficient of 0.49 (P 3 0.003) was found. Contrary to coronary angiographic-based reports, this IVUS study revealed a significant atheromatous plaque burden at the culprit lesion of MI-related arteries as well as diffuse atherosclerosis in the reference segments proximal and distal to the lesion. The detection of compensatory enlargement may explain the discrepancies between the histopathological and the angiographic studies.     

Histochemistry of myelin. VIII. Proteolytic activity around multiple sclerosis plaques

Synopsis Twenty-two plaques from six cases of multiple sclerosis were examined histochemically, with particular reference to demonstrating proteolytic activity by the gelatin-silver autogram method. Proteolytic activity was increased around plaques that were characterized as active (i.e. with increased cell population and NADH dehydrogenase activity at their edges). Inactive plaques showed little or no proteolytic activity. Proteolytic activity around active lesions could not be attributed to lipid-laden microglia: oligodendroglia or the myelin sheath are considered as alternative sources. Acid phosphatase and non-specific cholinesterase were mainly localized in lipid-laden microglia and the activity of these two enzymes was not otherwise prominent around active plaques. The oligodendroglia from unaffected white matter were found not to contain either of these enzymes. In view of previous observations that proteolytic enzymes causein vitro myelin breakdown and are also implicated in Wallerian degeneration, it is suggested that local proteolysis may be an initiating factor in the demyelinating process of multiple sclerosis.Research Associate supported by the British Multiple Sclerosis Society     

Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell-cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5-1 h) in a conspicuous belt-like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA-treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle-containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane-detached vinculin-rich material from the zonula adhaerens . The experiments show that, upon release from their junction-mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt-like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half-desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective effect of melatonin on 3-NP induced striatal interneuron injury in rats

To confirm the effect of melatonin on 3-nitropropionic acid (3-NP)-induced striatal interneuron injury in rats, behavioral test, histology, immunohistochemistry and Western blotting were respectively used to characterize the behavioral changes of experimental animals in motor and cognition, the morphological changes of striatal interneurons and the expression level of protein markers induced by 3-NP. The results showed that (1) 3-NP induced dysfunction of experimental animals in movement, motor coordination and cognition could be relieved by melatonin treatment; (2) The 3-NP-induced lesion area was unvaryingly in dorsolateral striatum, with almost all neuronal loss in the lesion core, however, lots of neurons survived after melatonin treatment; (3) Immunohistochemical staining of the four interneuron types (parvalbuminergic, cholinergic, calretinergic, and neuropeptide Y-neuronal nitric oxide synthase co-containing) showed that, in the lesion core of 3-NP group, loss of the four interneuron types was obvious, but in transition zone, the processes and varicosities of calretinergic, and neuropeptide Y- neuronal nitric oxide synthase co-containing interneurons increased significantly. Melatonin treatment reduced the loss of the four interneuron types in the lesion core, and inhibited the increase of processes and varicosities in the transition zone; (4) Consistent with above results, the expression level of five interneuron protein markers were significantly increased in the striatum after melatonin treatment. Notably, in both the transition zone and the lesion core induced by 3-NP, TUNEL-positive cells were detected, but decreased significantly after melatonin treatment. The present results indicate that melatonin effectively protects the striatal neurons against the injury induced by 3-NP in rats.     

TRANSIENT LOCALIZATION OF γ-TUBULIN AROUND THE CENTRIOLES IN THE NUCLEAR DIVISION OF BOERGESENIA FORBESII (SIPHONOCLADALES, CHLOROPHYTA)

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.