[Ecthyma gangrenosum associated with infection involving a methicillin-sensitive, Panton-Valentine-negative strain of Staphylococcus aureus]. / Ecthyma gangréneux dû à une souche de Staphylococcus aureus sensible à la méthicilline et non sécrétrice de toxine de Panton-Valentine.

BACKGROUND: Ecthyma gangrenosum (EG) is an anatomoclinical syndrome commonly associated with Pseudomonas aeruginosa cutaneous infection. Other microorganisms have also been incriminated on occasion, with other viral, fungal and bacterial agents potentially causing EG. In this report, we present an extremely rare case of an EG caused by methicillin-sensitive Staphylococcus aureus (MSSA) infection. This case, highly characteristic of EG both clinically and histologically, calls into question the physiopathological mechanisms of the disease and provides a reminder that it may be caused by a variety of organisms. PATIENTS AND METHODS: A 62-year-old woman, followed for HIV seropositivity at the AIDS stage, developed a painful purpuric skin rash evolving towards necrotic nodules characteristic of ecthyma gangrenosum. Skin biopsy confirmed the diagnosis of EG due to methicillin-sensitive S. aureus (MSSA) infection without toxins or bacteraemia. DISCUSSION: To the best of our knowledge, this is the first case in the literature in which MSSA is reported as the underlying cause of such lesions.

Role of the lean body mass and of pharmacogenetic variants on the pharmacokinetics and pharmacodynamics of sunitinib in cancer patients.

INTRODUCTION: Sunitinib is a multikinase inhibitor active in various cancers types including renal cancers and endocrine tumors. The study analyzed the influence of the lean body mass (LBM) and of pharmacogenetic variants on the exposure to sunitinib and its active metabolite, SU12662, and on sunitinib toxicity and clinical activity. MATERIALS AND METHODS: Exposure to sunitinib and SU12662 was assessed on days 10 and 21 during the first treatment cycle. Acute toxicity was graded using the NCI 4.0 CTCAE ver. 4.0. The LBM and 14 common single nucleotide polymorphisms in the CYP3A4/3A5, NR1I2, NR1I3, ABCB1, and ABCG2 genes were analyzed according to the drug exposure at day 10. Determinants (including sunitinib exposure and pharmacogenetic variants) for toxicities were assessed, as well as the relationship between drug exposure and survival in renal cancer patients. RESULTS: Ninety-two patients (60 % with renal cancer) were assessable for pharmacokinetics, toxicity and survival, and 66 for genetic analysis. The LBM (p < 0.0001) and a polymorphism in the ABCG2 transporter (421C>A) (p = 0.014) were two independent parameters accounting for the variability of composite (sunitinib + SU12662) exposure. Advanced age (OR = 1.47 [1.01-2.15], p = 0.048) and high sunitinib exposure (OR = 1.16 [1.05-1.28], p = 0.005) were independently associated with any grade ≥ 3 acute toxicity, and high SU12662 exposure was associated with grade ≥ 2 thrombocytopenia (OR = 1.27 [1.03-1.57], p = 0.028). A high composite area under the curve (AUC) >1,973 ng/mL∙h at day 21 was associated with a doubled survival (35.2 vs 16.7 months; log-rank p = 0.0051) in renal cancer patients. CONCLUSIONS: This study indicates that LBM and drug monitoring may be helpful in the management of sunitinib-treated patients.

Autochthonous falciparum malaria possibly transmitted by luggage-carried vector in Paris, France, February 2013.

We report a case of autochthonous falciparum malaria in a patient in Paris, France, in February 2013 who reported no recent travel to malaria-endemic countries. The parasite, Plasmodium falciparum, was possibly transmitted by an infective Anopheles mosquito carried in baggage from a malaria-endemic area.

[Management of candidemia and invasive candidiasis]. / Prise en charge des infections systémiques àCandida spp.

Candida species is the fourth most common cause of bloodstream infection and is the leading cause of invasive fungal infection among hospitalized patients. Acute disseminated candidiasis remains a life-threatening disease that now occurs mainly in intensive care units hospitalized patients. Delay in treatment of Candida bloodstream infections could be minimized by the development of more rapid and sensitive diagnostic techniques for the identification of Candida bloodstream infections. Current guidelines for the management of invasive candidiasis recommend fluconazole or an echinocandin as the primary therapeutic option. The optimal choice of the antifungal agent should depend on local epidemiology, prior antifungal therapy and patient's characteristics.

[Emergence of Candida parapsilosis candidemia at Cochin hospital. Characterization of isolates and search for risk factors]. / Émergence de candidémies à Candida parapsilosis à l'hôpital Cochin. Caractérisation des isolats et recherche de facteurs de risque.

Candida parapsilosis is a normal saprophyte of the skin, characterized by their affinity for catheters. This species has, in vitro, a level of sensitivity against the echinocandins, significantly lower than that observed with other Candida species. Recently, new species: Candida orthopsilosis and Candida metapsilosis, phenotypically identical to C. parapsilosis, have been identified by molecular biology. From 2003 to 2007, in the Cochin hospital, the proportion of C. parapsilosis among non-albicans species isolated from blood cultures increased from 17 (3/18) to 38% (5/13). To understand the reasons for this emergence, we retrospectively characterized isolates, conducted a case-control and researched a link between the emergence and introduction of caspofungin in our hospital. We analysed data from 26 patients who had candidemia with C. parapsilosis. Genotypic analysis of isolates has not identified the new species C. orthopsilosis and C. metapsilosis. The case-control study showed a broad-spectrum antibiotics was significantly more frequent for candidemia with C. parapsilosis compared to C. albicans (52 versus 26%, P=0.04) as a previous treatment with caspofungin (11 versus 0%, P=0.04). The introduction of caspofungin is contemporary with the emergence of candidemia with C. parapsilosis with a tendency to be related to its level of consumption in the ICU. Our results should encourage biologists to closely monitor the frequency and level of sensitivity of strains of C. parapsilosis isolated in hospital.

[Epidemiology of patients harboring extended-spectrum beta-lactamase-producing enterobacteriaceae (ESBLE), on admission]. / Épidémiologie des patients porteurs d'entérobactéries sécrétrices de bêtalactamase à spectre élargi (EBLSE), à l'admission.

OBJECTIVE: The incidence of extended-spectrum beta-lactamase-producing enterobacteria (ESBLE) has regularly increased over the last few years. However, little is known about epidemiology of ESBLE carriers in France. The objective of this study was to determine the ESBLE carriers or infected patients profile, identified within 48 hours following hospital admission. DESIGN: This retrospective study included all patients admitted in 2006 and 2007 at the Necker-Enfants-Malades (NEM) teaching hospital, carrying or infected with ESBLE isolated within 48 hours following admission. The pediatric and adult populations were compared. RESULTS: There was no significant difference between pediatric and adult populations. Escherichia coli and Klebsiella pneumoniae were the two main species isolated, accounting respectively for 59.6 and 21.1 % of the 114 isolated strains. Among the 114 analyzed files, 24 patients (21 %) were known to be EBLSE carriers, 37 (32 %) were transferred from another hospital, including 16 from another country. Concerning the 54 (47 %) other patients, five (4 %) came from a country with high prevalence, and 44 (39 %) were treated for a chronic illness. Only five patients (4 %) carrying ESBLE did not have any usual risk factor for multidrug resistance (MDR) bacterial carriage. CONCLUSIONS: In our study, 4 % of patients carrying ESBLE admitted had no usual risk factor for MDR bacteria. Targeted screening of previous carriers, patients with chronic illness, transferred patients, or patients coming from country with high prevalence, would help to limit the spread of ESBLE.

[Optimized clinical use of vancomycin, a prospective observational study in a Paris teaching hospital]. / Optimisation de la prescription de la vancomycine : étude prospective observationnelle dans un centre hospitalo-universitaire parisien.

INTRODUCTION: Vancomycin is still the cornerstone of antibiotic therapy for patients with suspected or proven invasive methicillin resistant Staphylococcus aureus infections. However, clinical and pharmacodynamic studies underline that appropriate doses depend on the infection site, the patient's weight, his renal function, and the bacterial susceptibility. OBJECTIVE AND METHOD: In this prospective study made in a Paris teaching hospital, our two goals were to describe the modalities of infusion and serum concentration obtained during therapy, in our pediatrics and adults population. RESULTS: In our hospital, vancomycin was administered every eight hours in 83 % (97/102) of the cases and the doses used were 30 mg/kg per day in 67 % of cases (68/102). Serum trough levels reached 15 mcg/ml and 20 mcg/ml in 36 % and 18 % of cases respectively. Moreover, despite adequate doses, trough levels of 15 mcg/ml were obtained in only 40 % of cases. CONCLUSION: Vancomycin infusion use could be optimized, by defining optimal serum concentrations and monitoring made by a mobile team of infectious diseases specialists.

The pathway of MAP kinase mediation of CSF arrest in Xenopus oocytes.

A cytoplasmic activity in mature oocytes responsible for second meiotic metaphase arrest was identified over 30 years ago in amphibian oocytes. In Xenopus oocytes CSF activity is initiated by the progesterone-dependent synthesis of Mos, a MAPK kinase kinase, which activates the MAPK pathway. CSF arrest is mediated by a sole MAPK target, the protein kinase p90Rsk which leads to inhibition of cyclin B degradation by the anaphase-promoting complex. Rsk phosphorylates and activates the Bub1 protein kinase, which may cause metaphase arrest due to inhibition of the anaphase-promoting complex (APC) by a conserved mechanism defined genetically in yeast and mammalian cells. CSF arrest in vertebrate oocytes by p90Rsk provides a potential link between the MAPK pathway and the spindle assembly checkpoint in the cell cycle.

Cell cycle transitions in early Xenopus development.

Xenopus oocytes and embryos undergo two major maternally controlled cell-cycle transitions: oocyte maturation and the mid-blastula transition (MBT). During maturation, the essential order of events in the cell cycle is perturbed in that the M phases of Meiosis I and II occur consecutively without an intervening S phase. Use of U0126, a new potent inhibitor of MAPK kinase (MEK), shows that MAPK activation is essential to inhibit the anaphase-promoting complex and cyclin B degradation at the MI/MII transition. If MAPK is inactivated, cyclin B is degraded, S phase commences and meiotic spindles do not form. These events are restored in U0126-treated oocytes by a constitutively active form of the protein kinase p90Rsk. Thus all actions of MAPK during maturation are mediated solely by activation of p90Rsk. At the MBT, commencing with the 13th cleavage division, there are profound changes in the cell cycle. MBT events such as maternal cyclin E degradation and sensitivity to apoptosis are regulated by a developmental timer insensitive to inhibition of DNA, RNA or protein synthesis. Other events, such as zygotic transcription and the DNA replication checkpoint, are controlled by the nuclear:cytoplasmic ratio. Lengthening of the cell cycle at the MBT is caused by increased Tyr15 phosphorylation of Cdc2 resulting from degradation of the maternal phosphatase Cdc25A and continued expression of maternal Wee1. Ionizing radiation causes activation of a checkpoint mediating apoptosis when administered before but not after the MBT. Resistance to apoptosis is associated with increased p27Xic1, the relative fraction of Bcl-2 or Bax in pro- versus anti-apoptotic complexes, and the activity of the protein kinase Akt.

Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes.

Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.

The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes.

In the Xenopus oocyte system mitogen treatment triggers the G(2)/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21(Cip1) had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.

Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation.

BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).

The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk).

BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.

Human retinoblastoma protein (Rb) is phosphorylated by cdc2 kinase and MAP kinase in Xenopus maturing oocytes.

Xenopus oocyte meiotic maturation combines features of G0/G1 and G2/M transitions of the cell cycle. To study the in ovo Rb kinase activity, we have microinjected human Rb into oocytes. Microinjected human Rb localizes into the nucleus, is hypophosphorylated in prophase oocytes, becomes hyperphosphorylated during meiotic maturation and is dephosphorylated as the cell reenters interphase. Inactivation or overexpression of the cyclin D-cdk4/6 complex in an oocyte extract does not affect the Rb kinase activity. This kinase activity could be attributed to both cdc2-cyclin B and MAP kinase, opening new perspectives of investigation in somatic cells.

On cyclins, oocytes, and eggs.

Oocyte and egg are suitable model systems for studying cell division since meiotic maturation resembles a G2/M transition and early embryonic divisions are precisely timed and occur without zygotic transcription. The analysis of oocytes and eggs from different species provides the opportunity to understand the roles of proteins that the critical to the progression and maintenance of the cell cycle. Among them, cyclins are certainly worthy of investigation. Mitotic cyclins (cyclins A and B) are clearly implicated in meiosis and early embryonic cell cycles. More recent studies have revealed that G1-type cyclins (cyclins E and D) could also play a role in both processes and cyclin H has been suggesed to participate to CAK activity (cdc2-activating kinase) in oocytes. The study of cyclins in oocytes and eggs clearly offer insights into their roles during the cell cycle.

Cyclin D2 arrests Xenopus early embryonic cell cycles.

Xenopus cyclin D2 mRNA is a member of the class of maternal RNAs. It is rare and stable during early embryonic development. To investigate the potential role of cyclin D2 during early embryonic cell cycles, cyclin D2 was injected into one blastomere of a two-cell embryo. This injection induced a cell cycle arrest in the injected blastomere. To analyze more precisely the mechanism of this arrest, we took advantage of cycling egg extracts that recapitulate major events of the cell cycle when supplemented with demembranated sperm heads. When Xenopus cyclin D2 is added to egg extracts, the first round of DNA replication occurs as in control extracts. However, Xenopus cyclin D2 blocks subsequent rounds of DNA replication and the oscillations of histone H1 kinase activity associated with cdc2 kinase, indicating that the cell cycle is arrested after the first S-phase. The block induced by Xenopus cyclin D2 is not due to a lack of the mitotic cyclin B2 that accumulates normally. Radiolabeled Xenopus cyclin D2 enters nuclei after completion of the first S-phase and remains stable over the entire period of the arrest. These features suggest that Xenopus cyclin D2 could play an original role during early development, controlling the G2-phase and/or the G2/M transition.

Increased stable retroviral gene transfer in early hematopoietic progenitors released from quiescence.

It has been previously demonstrated that prestimulation with cytokines could improve gene transfer in hematopoietic progenitors. However, we have shown that no combination of cytokines so far tested is able to release rapidly in vitro the stem cell compartment from quiescence unless an autocrine transforming growth factor-beta 1 (TGF-beta 1) is blocked by specific oligonucleotide antisense or antiserum (Hatzfeld et al., 1991, J. Exp. Med., 174, 925). We now report that a 10-hr cytokine prestimulation of SBA-CD34high human umbilical cord blood progenitors increases retrovirally mediated transfer of the nls-lacZ reporter gene from 1% to 23.8% and addition of anti-TGF-beta serum doubles this increase (47.3%). Interestingly, the effect of anti-TGF-beta preincubation on gene transfer is most effective on the most immature progenitors, which develop into high proliferative potential mixed colonies with 1-2 x 10(5) cells. Anti-TGF-beta serum pretreatment increases gene transfer in these early colony-forming units granulocyte-erythroid-megakaryocyte-macrophage (CFU-GEMM) from 54.1% to 93.3%. It augments significantly the stability of gene expression in all subpopulations of mixed colonies. Colonies obtained after pretreatment with anti-TGF-beta serum are larger and the expression of the stably integrated recombinant provirus does not reduce their size. This prestimulation method provides a substantial improvement for gene transfer efficiency within the quiescent stem cell compartment that is responsible for long-term engraftment.

Xenopus cyclin D2: cloning and expression in oocytes and during early development.

We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis cyclin D2 protein. Cyclin D2 mRNA is identified as a member of the class of maternal RNAs. It is rare and stable during embryonic development at least until tadepole. In addition, a second cDNA coding for a truncated version of cyclin D2 was also isolated. Microinjection of cyclin D2 into oocytes undergoing meiotic maturation and parthenogenetic activation reveals that the protein is stable for several hours, independently of the ubiquitin-mediated degradation of cyclin B2 that takes place periodically during this process. Microinjected cyclin D2 localizes both in the cytoplasm and in the nucleus of oocyte. In somatic cells, it is well established that cyclin D2 is almost exclusively nuclear and very labile. The unusual behaviour of cyclin D2 upon injection into oocytes may provide indications about a possible role for this protein during meiosis and early development.

Association of p34cdc2 kinase and MAP kinase with microtubules during the meiotic maturation of Xenopus oocytes.

p34cdc2 protein is found in prophase, metaphase and activated Xenopus oocytes at a similar level whereas its kinase activity oscillates within meiosis. Using an anti-PSTAIRE antibody that recognizes Xenopus p34cdc2, it was demonstrated that the major part of p34cdc2 was associated with microtubules isolated in vitro from Xenopus oocytes. Conversely, tubulin was recovered in association with p34cdc2 in p13-Sepharose pellets. The abundance of the fraction of p34cdc2 which was associated with microtubules did not oscillate during the meiotic maturation and the activation process. By contrast, the histone H1 kinase activity of p34cdc2 estimated in microtubular oocyte pellets was much higher in metaphase than in prophase oocytes. Cyclin B, which is associated in vivo with p34cdc2 in prophase and metaphase oocytes, was also present in the microtubular fractions. However, cyclin was not necessary for the binding of p34cdc2 to microtubules since p34cdc2 from activated eggs, where cyclin was missing, still copurified with microtubules. Purified MAP2, but not tubulin, was able to bind to p34cdc2, demonstrating that the association between p34cdc2 and microtubules was mediated by microtubule-associated proteins. During the meiotic maturation of Xenopus oocytes, several protein kinases were activated, among them MAP kinase. MAP kinase also associated with microtubules. It was demonstrated that both p34cdc2 kinase and MAP kinase purified from Xenopus oocytes were able to phosphorylate in vitro rat brain MAP2. However both protein kinases phosphorylated different domains of MAP2, suggesting that they might regulate microtubules in different ways.