Nightlife clusters of coronavirus disease in Tokyo between March and April 2020.

We analysed associations between exposure to nightlife businesses and severe acute respiratory syndrome coronavirus 2 PCR test results at a tertiary hospital in Tokyo between March and April 2020. A nightlife group was defined as those who had worked at or visited the businesses. We included 1517 individuals; 196 (12.9%) were categorised as the nightlife group. After propensity score matching, the proportion of positive PCR tests in the nightlife group was significantly higher than that in the non-nightlife group (nightlife, 63.8%; non-nightlife, 23.0%; P < 0.001). An inclusive approach to mitigate risks related to the businesses needs to be identified.

Development and validation of the comprehensive assessment scale for chemotherapy-induced peripheral neuropathy in survivors of cancer.

BACKGROUND: Appropriate assessment is essential for the management of chemotherapy-induced peripheral neuropathy (CIPN), an intractable symptom that cannot yet be palliated, which is high on the list of causes of distress for cancer patients. However, objective assessment by medical staff makes it easy to underestimate the symptoms and effects of CIPN in cancer survivors. As a result, divergence from subjective evaluation of cancer survivors is a significant problem. Therefore, there is an urgent need to develop a subjective scale with high accuracy and applicability that reflects the experiences of cancer patients. We developed a comprehensive assessment scale for CIPN in cancer survivors, named the Comprehensive Assessment Scale for Chemotherapy-Induced Peripheral Neuropathy in Survivors of Cancer (CAS-CIPN), and demonstrated its reliability and validity. METHODS: We developed a questionnaire based on qualitative studies of peripheral neuropathy in Japanese cancer patients and literature review. Twelve cancer experts confirmed the content validity of the questionnaire. A draft version comprising 40 items was finalized by a pilot test on 100 subjects. The participants in the present study were 327 Japanese cancer survivors. Construct validity was determined by factor analysis, and internal validity by confirmation factor analysis and Cronbach's α. RESULTS: Factor analysis showed that the structure consisted of 15 items in four dimensions: "Threatened interference in daily life by negative feelings", "Impaired hand fine motor skills", "Confidence in choice of treatment/management," and "Dysesthesia of the palms and soles." The CAS-CIPN internal consistency reliability was 0.826, and the reliability coefficient calculated using the Spearman-Brown formula [q = 2r/(1 + r)] was 0.713, confirming high internal consistency and stability. Scores on this scale were strongly correlated with Gynecologic Oncology Group-Neurotoxicity scores (r = 0.714, p < 0.01), confirming its criterion-related validity. CONCLUSIONS: The CAS-CIPN is an assessment tool with high reliability and validity for the comprehensive evaluation of CIPN in cancer survivors. The CAS-CIPN is simple to use, and can be used by medical professionals for appropriate situational assessment and intervention.

Involvement of interleukin-1 in lipopolysaccaride-induced microglial activation and learning and memory deficits.

We have developed an animal model of learning and memory impairment associated with activation of microglia in the mouse brain. Injection of lipopolysaccharide into the CA1 region of the mouse hippocampus resulted in an increased production of inflammatory cytokines, such as interleukin-1ß. Immunostaining for interleukin-1ß revealed an increase in the signal at 6 hr after lipopolysaccharide injection. Immunopositive cells for interleukin-1ß were colocalized with those immunopositive for CD11b. When subacute lipopolysaccharide treatment (20 µg/2 µl/injection, bilaterally for 5 consecutive days) was performed, long-term activation of microglia and learning and memory deficits as evaluated using a step-through passive avoidance test were observed in the wild-type mice. Gene expression of the N-methyl-D-aspartate receptor NR1 and NR2A subunits was also decreased by the lipopolysaccharide treatment. In contrast, activation of microglia and the associated behavioral deficits were not observed in mice lacking interleukin-1α and -1ß following the subacute lipopolysaccharide treatment, together with little change in the gene expression of NR1 and NR2A subunits. However, the subacute lipopolysaccharide treatment produced almost similar changes in those parameters in the tumor necrosis factor-α knockout mice as in the wild-type animals. The injection of interleukin-1ß neutralizing antibody with lipopolysaccharide for 5 consecutive days resulted in the improvement of lipopolysaccharide-induced learning and memory deficits. These findings suggest that the expression of interleukin-1 plays an important role in lipopolysaccharide-induced activation of microglia and the associated functional deficits in learning and memory.

The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1.

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular analysis of digenic inheritance in Bartter syndrome with sensorineural deafness.

BACKGROUND: Bartter syndrome (BS) is a genetic disorder accompanied by hypokalaemic metabolic alkalosis. BS with sensorineural deafness (SND, OMIM602522) is a newly identified phenotype caused by mutations in the BSND gene that encodes barttin, a beta-subunit for chloride channel ClC-Ka and ClC-Kb and classified as type IV BS. Type IV BS features the most severe phenotype entailing life-threatening neonatal volume depletion and chronic renal failure developing during infancy. A recent report described a case of BS with SND from a consanguineous family who showed homozygous mutations in the CLCNKA and CLCNKB genes. This case indicated the possibility of the occurrence of digenic inheritance in BS with SND resulting from double mutations in the CLCNKA and CLCNKB genes. SUBJECT AND RESULTS: The current report concerns a 2-year-old girl from a non-consanguineous family with BS accompanied by SND. In our case, four loss-of-function mutations, consisting of mutations in both parental alleles in both CLCNKA and CLCNKB, were identified. The paternal allele had a nonsense mutation (Q260X) in CLCNKA and a splicing site mutation (IVS17+1 g>a) in CLCNKB. The maternal allele had a large deletion mutation (about 12 kbp) extending from CLCNKA to CLCNKB. Our case provides clear evidence that loss-of-function alleles in both alleles of both CLCNKA and CLCNKB results in a phenotype indistinguishable from that of mutations in BSND (type IV BS). CONCLUSIONS: Recent advances in genetics have resulted in a better understanding of many human inherited diseases, but most of them are monogenic disorders and more complex inheritance patterns remain unresolved. Our case provides clear evidence of digenic inheritance outside the scope of Mendelian inheritance disorders.

Alpha-actinins, calspectin (brain spectrin or fodrin), and actin participate in adhesion and movement of growth cones.

We have used biochemical and immunocytochemical techniques to investigate the possible involvement of membrane cytoskeletal elements such as alpha-actinin, calspectin (brain spectrin or fodrin), and actin in growth cone activities. During NGF-induced differentiation of PC12 cells, alpha-actinin increased in association with neurite outgrowth and was predominantly distributed throughout the entire growth cone and the distal portion of neurites. Filopodial movements were sensitive to Ca2+ flux. Two types of alpha-actinin, with Ca2(+)-sensitive and -insensitive actin binding abilities, were identified in the differentiated cells. Ca2(+)-sensitive alpha-actinin and actin filaments were concentrated in filopodia. The Ca2(+)-insensitive protein was distributed from the body of the growth cone to the distal portion of neurites, corresponding to the substratum-adhesive sites. The location of calspectin in growth cones was similar to that of the Ca2(+)-insensitive alpha-actinin. These results are consistent with the hypothesis that Ca2(+)-sensitive alpha-actinin and actin filaments are involved in Ca2(+)-dependent filopodial movement and Ca2(+)-insensitive alpha-actinin and calspectin are associated with adhesion of growth cones.

Activation of the c-myc p1 promoter in Burkitt's lymphoma by the hs3 immunoglobulin heavy-chain gene enhancer.

The expression of c-myc is deregulated in Burkitt's lymphoma by the translocation t(8;14). Most of the increased c-myc expression is from the P1 promoter, which is normally a minor promoter. How the P1 promoter is activated by the immunoglobulin heavy chain gene enhancers is not understood. We identified a YY1 site in the immunoglobulin heavy-chain gene HS3 enhancer, which increased c-myc P1 promoter activity, and a MARE site, which decreased c-myc P1 activity. Small Maf proteins bound to the MARE site both in vitro and in vivo, recruited histone deacetylase 2, and resulted in deacetylation of histones H3 and H4 at the c-myc promoter region. In contrast, YY1 recruited CBP and increased histone acetylation at the c-myc promoter. Rb interacts with YY1 to prevent DNA binding in normal B cells, but no significant interaction with YY1 was detected in Burkitt's cells, and binding of YY1 to the HS3 enhancer was observed by chromatin immunoprecipitaton. Increased expression of MafK and/or decreased expression of YY1 by silencing RNA downregulated endogenous c-myc mRNA levels and increased the sensitivity of the cells to doxorubicin. Mutation of the major active sites (nuclear factor-kappa B and YY1) in the enhancers prevented c-myc activation.

Estimating nurses' workload using the Diagnosis Procedure Combination in Japan.

AIM: To examine the methods used to estimate nurse staffing levels in acute care settings with Diagnosis Related Groups, which in Japan are called the Diagnosis Procedure Combination (DPC). METHODS: For estimating staffing requirements, the study used four DPC groups: (1) acute or recurrent myocardial infarction (AMI) with stenting, (2) angina pectoris with coronary artery bypass grafting (CABG), (3) sub-arachnoid haemorrhage (SAH) with clipping surgery, and (4) cerebral infarction with carotid endarterectomy (CEA). Registered nurses with more than 3-year nursing experience in nine university hospitals in the Tokyo metropolitan area completed self-report questionnaires in order to obtain nursing care time and care intensity per each DPC. The concordance rate was measured by Kendall's coefficient of concordance. The relationship between the care time and the care intensity was examined by a time series graph per DPC. Care intensity consisted of professional judgement, mental effort for helping patients, professional skill, physical effort for providing activities of daily living support, and nurse stress, based on the Hsiao and colleagues' model of resource-based relative value scale. RESULTS: Twenty-five nurses in nine university hospitals answered for a hypothetical typical patient with AMI and with CABG, and 28 nurses in nine university hospitals answered for a hypothetical typical patient with SAH and with CEA. Kendall's coefficient of concordance was 0.896 for AMI, 0.855 for CABG, 0.848 for SAH, 0.854 for CEA. The time series data of the care time and the care intensity items showed different patterns for each DPC. CONCLUSION: The DPC for cardiovascular and cerebral surgical procedures can be used for estimating nurses' workload.

The EBNA2-related resistance towards alpha interferon (IFN-alpha) in Burkitt's lymphoma cells effects induction of IFN-induced genes but not the activation of transcription factor ISGF-3.

Transfection of a plasmid encoding the Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) gene confers resistance to the antiproliferative effect of alpha interferon (IFN-alpha) in EBV-negative U968 cells (P. Aman and A. von Gabain, EMBO J. 9:147-152, 1990). We studied the expression of IFN-stimulated genes (ISGs) in two pairs of Burkitt's lymphoma cell lines, differing in the expression of the putative immortalizing gene of EBV, EBNA2. In EBNA2-expressing cells, the induction of four ISGs by IFN-alpha was strongly reduced or, in some cases, abolished. Chloramphenicol acetyltransferase reporter gene constructs containing different IFN-stimulated response elements were transfected into EBNA2-negative and EBNA2-positive cells. Induction of chloramphenicol acetyltransferase activity by IFN was impaired in EBNA2-positive cells. Also, a reporter gene construct driven by an IFN-gamma-sensitive promoter element was affected. However, as revealed by gel shift assays, EBNA2-positive and EBNA2-negative cells exhibited a nearly identical pattern of IFN-stimulated response element-binding proteins. Most important, activation of the factor ISGF-3, which previously has been shown to be required and sufficient for transcriptional activation of IFN-induced genes, was not inhibited in IFN-resistant cells expressing EBNA2. The mechanism of the EBNA2-related IFN resistance seems to be distinct both from the resistance mediated by hepatitis virus and adenovirus gene products and from the IFN resistance in Daudi cell variants. In these three cases, the transcriptional block of IFN-induced genes is due to inhibition of ISGF-3 activation and binding. Our data suggest that the EBNA2-related IFN resistance in Burkitt's lymphoma cells acts downstream of the activation of ISGF-3.

Mammary carcinogenic effect of low-dose fission radiation in Wistar/Furth rats and its dependency on prolactin.

The mammary carcinogenic effect in rats of low-dose fission radiation and its dependency on prolactin were studied. A total of 141 female W/Fu rats were exposed to 4.8, 8.9, or 19.5 rads of fission radiation that had both fision neutrons of 2.0 million electron volts (MeV) and gamma ray components similar to those produced by the Hiroshima bomb. Only 1 of 48 rats (2.0%) developed mammary tumor (MT) after irradiation alone, whereas 20 of 48 rats (41.6%) developed MT's if prolactin was supplied shortly after irradiation by means of grafting of the prolactin-secreting pituitary tumor. Furthermore, MT's occurred in 11 of 45 rats (24.4%) treated with prolactin as late as 12 months after irradiation, which suggested the long-term survival of radiation-induced dormant MT cells. A correlation was found between the development of MT and the elevation of serum prolactin level; most MT's appeared shortly after the grafted mammotropic pituitary tumor became palpable. The growth of MT's appeared to be promoted by prolactin in collaboration with ovarian hormones; the growth of adenocarcinomas was dependent on prolactin and ovarian hormones, whereas the growth of fibroadenomas appeared to be less hormone-dependent. Much higher biologic effectiveness, especially in the low-dose range, was found with 2.0-MeV fission neutrons compared with 14.1-MeV fast neutrons or 180-kilovolt peak X-rays in rat mammary carcinogenesis.

Localization of 4.1 related proteins in cerebellar neurons.

Localization of 4.1 related proteins in neurons was studied with immunofluorescence microscopy and with immunoelectron microscopy on ultrathin cryosections. In rat cerebellum, 4.1 immunoreactive proteins were demonstrated in Purkinje cell bodies, dendrites and other neurons in the cerebellar cortex. Some glial cells showed staining, but no labeling was found in myelinated axons of the white matter and of the glomeruli in the granule cell layer. At the ultrastructural level, the 4.1 related proteins were localized mainly in the cytoplasmic matrix, while some labeling was found underneath the plasma membrane. To determine whether 4.1 related proteins in neuronal cytoplasm exist as part of the cytoskeleton or not, PC12 cells cultured in the presence of nerve growth factor were stained with the anti-4.1 antibody. Since cytoplasmic staining was retained after detergent treatment, the 4.1 related proteins seem to exist as a component of the neural cell cytoskeleton. Localization of 4.1 related proteins during the postnatal development of the cerebellum was also studied. In Purkinje cells, localization of 4.1 related proteins changed according to the stages of the postnatal development. The present data suggest that 4.1 related proteins in neurons localized mainly in the cytoplasm and may play some role in organizing cytoskeletal networks in the cytomatrix. Their distribution is developmentally regulated in some neurons, possibly in relationship to their maturation in the cytoskeleton.

Glucocorticoids potentiate the action of atrial natriuretic polypeptide in adrenalectomized rats.

Adrenal insufficiency in human and rat is associated with an impairment of the diuretic response to water load, and only glucorticoids (GCs) restore this deficit. Our observation that GCs potentiate atrial natriuretic polypeptide (ANP)-stimulated cGMP production in cultured renal cells prompted us to examine the possibility that GCs may restore the diuretic response through the potentiation of ANP action. Initially, changes in urine volume and ANP levels were studied in adrenalectomized (Adx) and sham-operated intact rats after an oral water load of 5 ml/100 g BW. Urine volume after water load was 4.5 +/- 0.5 ml/30 min in the intact rats, whereas it was 0.8 +/- 0.2 ml/30 min in the Adx rats. In the intact rats, a significant increase in plasma ANP level was observed 30 min after the water load, whereas no increase was observed in Adx rats. This defective ANP response may be involved in the impairment of the diuretic response in Adx rats. Indeed, pretreatment of Adx rats with dexamethasone (Dex, 20 micrograms/100 g BW) increased plasma ANP levels even before water load and improved diuretic response. Subsequently, effect of iv administration of human or rat ANP at a pharmacological dose (2.5 micrograms/100 g BW) on urine volume, osmolarity, and urinary excretion of cGMP, and sodium was studied in Adx rats that received an oral water load 30 min before ANP. Dex treatment was achieved by per os administration 3 h before the ANP injection. In Adx rats, the urine volume after ANP administration was 1.2 +/- 0.1 ml/30 min, and pretreatment with Dex markedly increased the urine volume to 6.3 +/- 0.4 ml/30 min. Dex also increased ANP-induced osmolar and sodium excretion by 2.6- and 2.9-fold, respectively. Although urinary excretion of cGMP was increased in Adx rats by ANP administration, a further significant increase was observed by the pretreatment with Dex. Injection of (Bu)2cGMP to Adx rats pretreated with Dex resulted in a significant increase in urine volume and osmolar and sodium excretion. However, no significant increase in urine volume was observed in Adx rats not pretreated with Dex. The present study suggests that GCs restore the diuretic response to acute water load not only by increasing the secretion of ANP but also by potentiating ANP-stimulated cGMP production. Furthermore, GCs may augment ANP action at one or more steps other than cGMP formation because administration of (Bu)2cGMP to Adx rats did not correct the diuretic response to water load.

A 3',5'-cyclic adenosine monophosphate-dependent pathway is responsible for a rapid increase in c-fos messenger ribonucleic acid by adrenocorticotropin.

ACTH rapidly and transiently increases c-fos mRNA in the rat adrenals in vivo. The present investigation was undertaken in order to determine what kind(s) of second messenger systems is involved in this increase. Rat adrenal cells were grown in monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. After 2 days of culture, cells were treated with ACTH and various agents alone or in combination. The amount of c-fos mRNA was determined by dot blot hybridization and corticosterone levels in the media were measured by RIA. ACTH (300 pg/ml) increased c-fos mRNA transiently with a peak level after 60 min. A similar increase was observed when (Bu)2cAMP (1 mM) was substituted for ACTH. Pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), a selective inhibitor of cAMP-dependent protein kinase, suppressed both basal and ACTH-increased c-fos mRNA. H-89 also suppressed corticosterone production. On the other hand, neither 12-O-tetradecanoyl-phorbol-13-acetate (100 ng/ml) nor elevated potassium ion (50 mM) affected the amount of c-fos mRNA and corticosterone production. Furthermore, pretreatment with cycloheximide (5 micrograms/ml) increased both basal and ACTH-increased c-fos mRNA. These results indicate that ACTH increases c-fos mRNA by phosphorylation of preexisting trans-acting factor(s) via cAMP-dependent protein kinase in common with steroidogenesis.

Parathyroid hormone-related peptide is a potent tumor angiogenic factor.

Rat pituitary malignant tumor cells; mGH3, show hypervascularization in in vivo xenografts and overexpress parathyroid hormone-related peptide (PTHrP) compared to original GH3 cells. To elucidate whether PTHrP is involved in tumor-derived angiogenesis, we examined the effect of PTHrP on vascular endothelial cells both in vitro and in vivo. Results of in vivo diffusion chamber assay showed a clear hypervascularization on the outer surface of diffusion chambers containing mGH3 tumor cell implants but not in those containing GH3 cells. Co-incubation with antisense PTHrP oligonucleotide (10 microM), but not sense or mismatched PTHrP oligonucleotide, suppressed hypervascularization in diffusion chambers. To further examine the role of PTHrP on endothelial cell function, PTHrP(1-34) was added at various concentrations to cultured bovine endothelial cells (BAECs) harvested from the aorta. PTHrP(1-34) did not alter the proliferation or migration of endothelial cells, but rather dose-dependently increased capillary formation by endothelial cells on the collagen gel matrix. Furthermore, 0.1 mM of 8-bromo-cAMP caused a similar increase in tube formation, which was dose-dependently inhibited by H89, a protein kinase A inhibitor. Our results indicate for the first time that PTHrP is a potential paracrine factor acting via the PKA pathway to enhance angiogenesis through capillary tube formation by endothelial cells in malignant pituitary tumors.

Recovery of motor-unit function after peripheral nerve injury in aged rats.

The potential capacity of aged motoneurons for the reconstruction of motor-units after nerve crush injury was studied in the medial gastrocnemius (MG) muscle of male Fischer rats. The MG nerve in middle-aged (8 months old) and aged (24 months) rats was aseptically crushed under pentobarbital anesthesia. After a 3-month recovery period, the animal was reanesthetized and physiological properties of individual motor-units were recorded. The three different types (fast twitch, fatigable: FF; fast twitch, fatigue resistant: FR and slow twitch: S) of normal motor-unit organization were restored in both middle-aged and aged reinnervated muscles as measured by their relative distributions, mean twitch contraction times and mean tetanic tensions. Some reinnervated units in both aged and middle-aged rats produced a large tetanic tension which exceeded the range for intact units. These findings indicate that aged motoneurons maintain their ability for axonal regenerating and muscle fiber innervation to reestablish normal function of motor-units.

Overloading a muscle does not alter the rate of motoneuronal loss in aged rats.

Effects of increased activity on neuronal cell death was investigated in the motor nuclei innervating normal and overloaded medial gastrocnemius (MG) muscles of Fischer 344 rats. The MG muscle was overloaded by the unilateral surgical ablation of synergists at the age of 17 months (group A) or 24 months (group B). When the rats reached the age of 24 and 28 months (group A) or 30 months (group B), motoneurons innervating the MG muscle were labeled bilaterally with horseradish peroxidase injected into the MG nerve. The wet weight of the muscle on the operated side was consistently heavier than that of the contralateral, intact side. The number of labeled neurons decreased with advancing age, and there was no difference in the magnitude of decline found between motor nuclei innervating intact and hypertrophied muscles. Thus, overloading the MG muscle did not retard or accelerate the age-related loss of motoneurons innervating this muscle. These findings indicate that the causal relationship between motoneuronal activity and death with advancing age needs to be studied further.

Role of Mg2+ in the ribozyme system.

Recently, several kinds of catalytic activities have been identified from L-19 IVS RNA or 'ribozyme'. In the catalytic process of ribozyme, Mg2+ is required. We investigated the role of Mg2+ by molecular orbital calculation. Based on the results of the calculation, a model for the self-splicing reaction was constructed.

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin. A spectrin-like calmodulin-binding protein (fodrin).

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated Mr of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca2+ and calmodulin. Peak II revealed a basal activity in the absence of Ca2+ and calmodulin, which was stimulated 2-fold by addition of Ca2+. Calmodulin had no effect on the peak II activity.

Actin polymerization induced by calspectin, a calmodulin-binding spectrin-like protein.

We have purified from a membrane fraction of bovine brain a calmodulin-binding protein (calspectin) that shares a number of properties with erythrocyte spectrin: It has a heterodimeric structure with Mr 240 000 and 235 000 and binds to (dimeric form) or crosslinks (tetrameric form) F-actin. We show that calspectin (tetramer) is capable of inducing the polymerization of G-actin to actin filaments by increasing nucleation under conditions where actin alone polymerizes at a much slower rate. Thus, brain calspectin behaves in the same manner as erythrocyte spectrin, supporting the idea that, in conjunction with actin oligomers it comprises the cytoskeletal meshwork underlying the cytoplasmic surface of the nerve cell.