Longitudinal changes in global structural brain connectivity and cognitive performance in former hospitalized COVID-19 survivors: an exploratory study.

BACKGROUND: Long-term sequelae of COVID-19 can result in reduced functionality of the central nervous system and substandard quality of life. Gaining insight into the recovery trajectory of admitted COVID-19 patients on their cognitive performance and global structural brain connectivity may allow a better understanding of the diseases' relevance. OBJECTIVES: To assess whole-brain structural connectivity in former non-intensive-care unit (ICU)- and ICU-admitted COVID-19 survivors over 2 months following hospital discharge and correlate structural connectivity measures to cognitive performance. METHODS: Participants underwent Magnetic Resonance Imaging brain scans and a cognitive test battery after hospital discharge to evaluate structural connectivity and cognitive performance. Multilevel models were constructed for each graph measure and cognitive test, assessing the groups' influence, time since discharge, and interactions. Linear regression models estimated whether the graph measurements affected cognitive measures and whether they differed between ICU and non-ICU patients. RESULTS: Six former ICU and six non-ICU patients completed the study. Across the various graph measures, the characteristic path length decreased over time (ß = 0.97, p = 0.006). We detected no group-level effects (ß = 1.07, p = 0.442) nor interaction effects (ß = 1.02, p = 0.220). Cognitive performance improved for both non-ICU and ICU COVID-19 survivors on four out of seven cognitive tests 2 months later (p < 0.05). CONCLUSION: Adverse effects of COVID-19 on brain functioning and structure abate over time. These results should be supported by future research including larger sample sizes, matched control groups of healthy non-infected individuals, and more extended follow-up periods.

Alterations in the fronto-limbic network and corpus callosum in borderline-personality disorder.

Neuroimaging research provides evidence of grey matter changes in the prefrontal-limbic network in borderline personality disorder (BPD), yet research scarcely examines the white matter (WM) within this circuitry. The present study aimed to explore WM in prefrontal-limbic brain networks within BPD. Quantitative diffusion tensor imaging (DTI-MRI) measures of fractional anisotropy (FA) and mean diffusion (MD) were used to analyze the neural pathways in fifteen individuals with BPD (M = 25, SD = 6.76), in comparison to thirteen healthy individuals (M = 27.92, SD = 8.41). Quantitative DTI-MRI measures of FA and MD were evaluated for the cingulum, the fornix, the corpus callosum (CC), the inferior longitudinal fasciculus (ILF), the superior longitudinal fasciculus (SLF) and the uncinate fasciculus (UF). Lower FA values for both the left and the right cingulum, the genu, body, and splenium of the CC, left ILF and right SLF were found in BPD, compared to healthy individuals. MD values were higher for the genu and splenium of the CC in BPD. The findings indicate that a large-scale emotional brain network is affected in BPD with alterations in MD and FA of WM prefrontal-limbic pathways of the heteromodal association cortex involved in emotion processing and emotion regulation.

The imaging findings of impingement syndromes of the lower limb.

In this article we provide an overview of impingement syndromes of the lower limb. At the level of the hip, femoroacetabular and ischiofemoral impingement are recognised. At the level of the knee, we discuss Hoffa's fat pad impingement, suprapatellar fat pad impingement, pericruciate impingement, and iliotibial band syndrome. The impingement syndromes associated with anterior cruciate ligament (ACL) repair and intercondylar osteophytes are also illustrated. Most impingement syndromes are described at the level of the ankle. These include, anterior, anterolateral, posterior, anteromedial, posterior, and posterolateral impingement. For these conditions, we describe the best technique and expected imaging findings. It should be kept in mind that many of these findings have been observed in the asymptomatic population. Impingement is essentially a clinical diagnosis and imaging findings should be considered as supportive elements for this clinical diagnosis.

Evidence of increased axillary blood flow velocity without increased handgrip strength and endurance in persons with a fibromuscular axillary arch.

BACKGROUND: The purpose of this in vivo study was to compare axillary artery blood flow velocity, and maximal handgrip strength and endurance performance in young subjects with and without an axillary arch (AA). MATERIALS AND METHODS: One hundred and fifty-six young adults were screened for the presence of an AA on their dominant arm side. After physical examination subjects were checked using diagnostic echography for the presence of an AA. Sixteen subjects with an AA and 15 without an AA had their axillary artery peak systolic velocity quantified in 3 different arm positions using Doppler ultrasound. Maximal handgrip strength and endurance performance was quantified in the same positions using a functional rehabilitation system. RESULTS: Mean peak systolic velocity was significantly higher in the AA group compared to controls in abduction/external rotation of the arm during muscle relaxation (p = 0.003) and contraction (p = 0.01). No significant differences between groups were found for maximal handgrip strength and endurance performance. CONCLUSIONS: This study provides evidence for a transient axillary artery compression by the AA in a throwing position. This is not reinforced by additional contraction of the shoulder muscles along with the AA. Axillary artery compression does not influence maximal handgrip strength and endurance performance in symptom-free young adults.

Differentiation between normal and metastatic lymph nodes in patients with skin melanoma: Preliminary findings using a DIXON-based whole-body MRI approach.

Purpose: Metastatic melanoma lymph nodes (MMLns) might be challenging to detect on MR-WBI, as both MMLns and normal lymph nodes (NLns) can show restricted water diffusion. Our purpose is to assess the potential contribution of the DIXON sequence in differentiating MMLns from NLns. Material and methods: We followed a cohort of 107 patients with stage IIIb/c and IV skin melanoma for 32 months using MR-WBI with DIXON, STIR, and DWI/ADC sequences. We compared signal intensity (SI) values of MMLns and NLns in the four series of the DIXON sequence (in/out-of-phase, fat_only, and water_only series). The fat fraction (SIfat_only/SIin) and the long:short axis ratio of MMLns were calculated. The fat fraction was also calculated in the fatty hila of NLns. Results: All MMLns (8 from 7 patients) showed SIout>SIin with a mean fat fraction of 10%. In 40 normal fatty hila (25 patients), the proportion of SIout>SIin was 100% and mean fat fraction was 89% (p<0.001 for fat fraction, Mann-Whitney U-test). In the cortex of NLns, a SIout>SIin pattern was identified in 41/113 cases from 19/40 patients. The median long:short axis ratio in MMLns was 1.13 (range 1.03-1.25). Conclusion: The combination of three features of MMLns (SIout>SIin, low-fat fraction and rounded shape) might hold promise in differentiating NLns from MMLns in patients with skin melanoma. Further research is warranted due to the small number of MMLns in our cohort.

MRI in hypertrophic mono- and polyneuropathies.

Different conditions that may lead to enlarged nerves or nerve roots include hereditary motor and sensory neuropathy (HMSN), neurofibromatosis (NF) type 1, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and intraneural perineurioma. Differential diagnosis of hypertrophic mono- and polyradiculopathies remains challenging but is important because of different treatments and prognosis. Magnetic resonance imaging (MRI) can identify the hypertrophic nerve segments and guide a fascicular biopsy. A fascicular biopsy will often be necessary for precise diagnosis.

Preclinical validation of the advection diffusion flow estimation method using computational patient specific coronary tree phantoms.

Coronary computed tomography angiography (CCTA) does not allow the quantification of reduced blood flow due to coronary artery disease (CAD). In response, numerical methods based on the CCTA image have been developed to compute coronary blood flow and assess the impact of disease. However to compute blood flow in the coronary arteries, numerical methods require specification of boundary conditions that are difficult to estimate accurately in a patient-specific manner. We describe herein a new noninvasive flow estimation method, called Advection Diffusion Flow Estimation (ADFE), to compute coronary artery flow from CCTA to use as boundary conditions for numerical models of coronary blood flow. ADFE uses image contrast variation along the tree-like structure to estimate flow in each vessel. For validating this method we used patient specific software phantoms on which the transport of contrast was simulated. This controlled validation setting enables a direct comparison between estimated flow and actual flow and a detailed investigation of factors affecting accuracy. A total of 10 CCTA image data sets were processed to extract all necessary information for simulating contrast transport. A spectral element method solver was used for computing the ground truth simulations with high accuracy. On this data set, the ADFE method showed a high correlation coefficient of 0.998 between estimated flow and the ground truth flow together with an average relative error of only 1 % . Comparing the ADFE method with the best method currently available (TAFE) for image-based blood flow estimation, which showed a correlation coefficient of 0.752 and average error of 20 % , it can be concluded that the ADFE method has the potential to significantly improve the quantification of coronary artery blood flow derived from contrast gradients in CCTA images.

Magnetic resonance imaging in the normal fetal heart and in congenital heart disease.

OBJECTIVE: To evaluate prospectively the feasibility of magnetic resonance imaging (MRI) for assessment of the fetal heart for congenital heart disease (CHD). METHODS: This was a cross-sectional study, including 66 fetuses with a normal heart and 40 with CHD. The fetal heart was examined on MRI using axial steady-state free precession (SSFP) sequences. Regression analysis was used to investigate the effect on the ability to visualize cardiac anatomy of gestational age at examination, maternal body mass index, presence of fetal cardiac abnormality, fetal movements, fetal lie and twinning. The sensitivity and specificity of detecting cardiac defects were calculated. RESULTS: The four-chamber view was visualized in 98.1% of fetuses. The sensitivity of detecting a cardiac defect on the four-chamber view was 88% and the specificity 96%. The ability to visualize the left and right outflow tracts was only influenced by the presence of fetal movements: for the left outflow tract 94.4 vs. 50.0% visualization and for the right outflow tract 92.6 vs. 53.8% visualization without and with fetal movements, respectively. The sensitivity of detecting a cardiac defect of the left outflow tract was 63% and the specificity 100%, while sensitivity and specificity were 59 and 97%, respectively, for the right outflow tract. CONCLUSIONS: Despite the use of SSFP sequences, MRI in the fetal heart remains of limited value. It can only be used as a second-line approach for abnormalities of the four-chamber view suspected at prenatal ultrasound.

Postmortem examination of human fetal hearts at or below 20 weeks' gestation: a comparison of high-field MRI at 9.4 T with lower-field MRI magnets and stereomicroscopic autopsy.

OBJECTIVES: To compare the diagnostic usefulness of high-field with low-field magnetic resonance imaging (MRI) and stereomicroscopic autopsy for examination of the heart in fetuses at or under 20 weeks' gestation. METHODS: Prior to invasive stereomicroscopic autopsy, MRI scans at 9.4, 3.0 and 1.5 T were performed on 24 fetuses between 11 and 20 weeks' gestation, including 10 fetuses with cardiac abnormalities. The ability to visualize different heart structures was evaluated according to the different field strength MRI magnets used and gestational age at examination. RESULTS: On 1.5- and 3.0-T MRI, only the heart situs and four-chamber view could be visualized consistently (in 75% or more of cases) when the fetus was beyond 16 weeks' gestation, but other heart structures could not be visualized for fetuses at any gestational age. In contrast, using high-field MRI at 9.4 T, the heart situs, four-chamber view and the outflow tracts could be visualized in all fetuses irrespective of gestational age. Using high-field MRI, the sensitivity for detecting an abnormality of the four-chamber view was 66.7% (95% CI, 30.1-92.1%) with a specificity of 80.0% (95% CI, 51.9-95.4%). For abnormalities of the outflow tracts, sensitivity was 75.0% (95% CI, 20.3-95.9%) and specificity 100.0% (95% CI, 83.3-100.0%). Eight fetuses out of 10 with congenital heart disease (CHD) were classified as having major CHD. High-field MRI at 9.4 T was able to identify seven out of the eight cases of major CHD. CONCLUSION: High-field MRI at 9.4 T seems to be an acceptable alternative approach to invasive stereomicroscopic autopsy for fetuses with CHD at or below 20 weeks' gestation.

Acceptance, reliability and confidence of diagnosis of fetal and neonatal virtuopsy compared with conventional autopsy: a prospective study.

OBJECTIVES: To compare prospectively maternal acceptance of fetal and neonatal virtuopsy with that of conventional autopsy and to determine the confidence with which magnetic resonance (MR) virtuopsy can be used to diagnose normality/abnormality of various fetal anatomical structures. METHODS: MR and/or computed tomography virtuopsy and conventional autopsy were offered to 96 women (102 fetuses/neonates) following termination of pregnancy (TOP), intrauterine fetal death (IUFD) or neonatal death. Multivariable logistic regression analysis was used to investigate the effect on maternal acceptance of virtuopsy and/or conventional autopsy of the age of the mother, gestational age at TOP or delivery after IUFD, order of pregnancy, parity, religion, type of caregiver obtaining consent and reason for death. When parents consented to both MR virtuopsy and conventional autopsy of fetuses ≥ 20 weeks of gestation or neonates, the confidence with which MR virtuopsy could be used to diagnose normality/abnormality of various anatomical structures was determined on a scale in which conventional autopsy was considered gold standard. On autopsy we classified fetuses/neonates as having either 'normal' or 'abnormal' anatomical structures; these groups were analyzed separately. At virtuopsy, we indicated confidence of diagnosis of normality/abnormality of every anatomical structure in each of these two groups defined at autopsy, using a scale from 0 (definitely abnormal) to 100 (definitely normal). RESULTS: Of the 96 women, 99% (n = 95) consented to virtuopsy and 61.5% (n = 59) to both conventional autopsy and virtuopsy; i.e. 36 (37.5%) consented to virtuopsy alone. Maternal acceptance of conventional autopsy was independently positively related to singleton pregnancy, non-Moslem mother, earlier gestation at TOP or delivery afer IUFD and a maternal-fetal medicine specialist obtaining consent. Thirty-three fetuses ≥ 20 weeks of gestation had both conventional autopsy and MR virtuopsy, of which 19 had a full autopsy including the brain. In fetuses with normal anatomical structures at conventional autopsy, MR virtuopsy was associated with high diagnostic confidence (scores > 80) for the brain, skeleton, thoracic organs except the heart, abdominal organs except the pancreas, ureters, bladder and genitals. In fetuses with abnormal anatomical structures at autopsy, MR virtuopsy detected the anomalies with high confidence (scores < 20) for these same anatomical structures. However, in three cases, virtuopsy diagnosed brain anomalies additional to those observed at conventional autopsy. CONCLUSION: MR virtuopsy is accepted by nearly all mothers while conventional autopsy is accepted by about two-thirds of mothers, in whom refusal depends mainly on factors over which we have no control. Although conventional autopsy remains the gold standard, the high acceptance of virtuopsy makes it an acceptable alternative when the former is declined.

Initial axillary staging of breast cancer using ultrasound-guided fine needle aspiration: a liquid-based cytology study.

OBJECTIVE: To evaluate the preoperative detection of axillary metastasis combining ultrasound (US)-guided fine needle aspiration cytology (FNAC) and liquid-based cytology (Surepath(®)) to reduce sentinel node procedures. METHODS: In total, 148 patients with clinically negative lymph nodes and no preoperative therapy were included. All patients underwent preoperative ultrasound of the axilla with FNAC if suspicious lymph nodes were found. Complete axillary lymph node dissection was performed at primary surgery when FNAC was positive. All other patients underwent a sentinel node procedure. RESULTS: US-guided FNAC of the axilla revealed metastasis in 34 (23.0%) of the 148 patients. These 34 patients were 53.1% of all patients (n = 64) with proven axillary lymph node involvement. In 66 patients (44.6%), both ultrasound and histopathology were negative. Overall sensitivity of US-guided FNAC was 50.0%, specificity 100%, positive predictive value 100% and negative predictive value 70.2%. In T1 tumours, all patients referred for sentinel node procedure were node-negative. The correlation between malignant FNAC and histopathology was 100%. US-guided liquid-based FNAC in patients with no clinically positive lymph nodes reduced the necessity for a sentinel node procedure by 23.0%. CONCLUSIONS: We advocate that US-guided fine needle aspiration (FNA) combined with liquid-based cytology of axillary lymph nodes should be included in the preoperative staging of breast cancer.

Magnetic resonance imaging of anatomical variants of the subtalar joint.

OBJECTIVE: To evaluate MRI with thin slices to depict anatomical variations of the subtalar and talocalcaneonavicular joints. METHODS AND MATERIALS: Ankle MRI was performed in 51 patients. The articular configurations were differentiated on sagittal T1-weighted and three-dimensional (3D) DESS images. Multiplanar reconstructions were performed. The variation in curvature of the posterior facet of the calcaneus and talus was analysed. 3D surface-rendered images of the calcaneus were obtained. All images were studied by an experienced physical therapist. RESULTS: Analysis revealed that 18 (36.7%) of the subjects had 3 distinct facets on the calcaneus. A missing anterior facet was revealed in five (10.2%) of subjects. Twenty-six (53.1%) subjects showed a fusion of the anterior and middle facets. In four cases, an articulation was found between the talus and the cuboid bone. At the posterior talocalcaneal articulation, variations were observed of the curvature of the medial side of the joint. CONCLUSIONS: The configuration of the facets of the talocalcaneal joints can be analysed on MR images. On 3D images, an articular connection was seen between the talus and the cuboid bone, as well as variations in the curvature of the posterior talocalcaneal joint. These anatomical variations may have implications for mobility and stability of the ankle.

Hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries is mediated by intracellular methylglyoxal levels in a pathway dependent on oxidative stress.

AIMS/HYPOTHESIS: Impaired nitric oxide (NO)-dependent vasorelaxation plays a key role in the development of diabetic vascular complications. We investigated the effect of hyperglycaemia on impaired vasoreactivity and a putative role therein of the AGE precursor methylglyoxal. METHODS: The effects of high glucose and methylglyoxal on NO-dependent vasorelaxation in isolated rat mesenteric arteries from wild-type and transgenic glyoxalase (GLO)-I (also known as GLO1) rats, i.e. the enzyme detoxifying methylglyoxal, were recorded in a wire myograph. AGE formation of the major methylglyoxal-adduct 5-hydro-5-methylimidazolone (MG-H1) was detected with an antibody against MG-H1 and quantified with ultra-performance liquid chromatography (tandem) mass spectrometry. Reactive oxygen species formation was measured with a 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester probe and by immunohistochemistry with an antibody against nitrotyrosine. RESULTS: High glucose and methylglyoxal exposure of mesenteric arteries significantly reduced the efficacy of NO-dependent vasorelaxation (p < 0.05). This impairment was not observed in mesenteric arteries of GLO-I transgenic rats indicating a specific intracellular methylglyoxal effect. The diabetes-induced impaired potency (pD(2)) in mesenteric arteries of wild-type rats was significantly improved by GLO-I overexpression (p < 0.05). Methylglyoxal-modified albumin did not affect NO-dependent vasorelaxation, while under the same conditions the receptor for AGE ligand S100b did (p < 0.05). Methylglyoxal treatment of arteries increased intracellular staining of MG-H1 in endothelial cells and adventitia by fivefold accompanied by an eightfold increase in the oxidative stress marker nitrotyrosine. Antioxidant pre-incubation prevented methylglyoxal-induced impairment of vasoreactivity. CONCLUSIONS/INTERPRETATION: These data show that hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation is mediated by increased intracellular methylglyoxal levels in a pathway dependent on oxidative stress.

Fast-release orodispersible tramadol as analgesia in hysterosalpingography with a metal cannula or a balloon catheter.

BACKGROUND: This study aimed to evaluate the potential benefit, in terms of pain relief, of the new oral fast-release orodispersible galvanic form of tramadol in women undergoing hysterosalpingography (HSG) with either a metal cannula or a balloon catheter. METHODS: In a randomized, double-blind, placebo-controlled, 2 x 2 factorial-design trial, conducted at a single academic centre, 128 women were assigned into groups: (I) tramadol and a metal cannula, (II) tramadol and a balloon catheter, (III) placebo and a metal cannula or (IV) placebo and a balloon catheter. The primary end-point was pain registered by the patients on 10-cm visual analogue scales (VASs) at various times during and after the procedure. Secondary end-points included side effects and pain as assessed by the same physician during HSG. RESULTS: The main effect of tramadol versus placebo medication (i.e. I and II versus III and IV) was a statistically significant difference (P < 0.001) in self-reported VAS of -0.91 (-1.35 to -0.47) on the absolute and -33% (-48% to -17%) on the relative scale in favour of tramadol. Likewise, there was a significant benefit for tramadol against placebo medication for physician-perceived VAS pain scores (39% relative reduction; P < 0.001). The main effect of the balloon catheter versus metal cannula (i.e. II and IV versus I and III) was a non-significant (P = 0.82) difference in patient-reported VAS of -0.05 (-0.49 to +0.39) and -2% (-21% to +17%). There were no medication-HSG device interactions and no differences in side effects. CONCLUSIONS: During and after HSG, fast-release orodispersible tramadol significantly reduces pain without increasing side effects.

Direct measurement of tibial cortical bone surface area.

Cortical bone surface area estimation is generally performed by analysis of medical images but its accuracy has not yet been verified by direct bone measurements. This study was performed to evaluate the reproducibility of direct ex vivo estimations of cross sectional tibial cortical bone surface area. Ten tibiae were evaluated by two observers using direct 3D-digitization at 38 and 66% of total tibial length using a 'critical' and a 'general' criterion for differentiation between cortical and cancellous bone. The results of the analysis using the more severe 'critical' differentiation criterion yielded high intra and interobserver agreement. Mean intraobserver reliability was demonstrated by R = 0.98 (Spearman rank correlation between 0.98 and 0.97; P < 0.01) and inter observer reliability by R = 0.91 (Spearman rank correlation between 0.95 and 0.88; P < 0.01). When using strict criteria to differentiate between cortical and cancellous bone, intra and interobserver reliability of the presented method of direct cortical bone surface area estimation is very high. The results indicate that cross-sectional cortical bone surface estimation by direct 3D-digitization can serve as a gold standard for validation of other estimations methods.

Synthetic MRI of Preterm Infants at Term-Equivalent Age: Evaluation of Diagnostic Image Quality and Automated Brain Volume Segmentation.

BACKGROUND AND PURPOSE: Neonatal MR imaging brain volume measurements can be used as biomarkers for long-term neurodevelopmental outcome, but quantitative volumetric MR imaging data are not usually available during routine radiologic evaluation. In the current study, the feasibility of automated quantitative brain volumetry and image reconstruction via synthetic MR imaging in very preterm infants was investigated. MATERIALS AND METHODS: Conventional and synthetic T1WIs and T2WIs from 111 very preterm infants were acquired at term-equivalent age. Overall image quality and artifacts of the conventional and synthetic images were rated on a 4-point scale. Legibility of anatomic structures and lesion conspicuity were assessed on a binary scale. Synthetic MR volumetry was compared with that generated via MANTiS, which is a neonatal tissue segmentation toolbox based on T2WI. RESULTS: Image quality was good or excellent for most conventional and synthetic images. The 2 methods did not differ significantly regarding image quality or diagnostic performance for focal and cystic WM lesions. Dice similarity coefficients had excellent overlap for intracranial volume (97.3%) and brain parenchymal volume (94.3%), and moderate overlap for CSF (75.6%). Bland-Altman plots demonstrated a small systematic bias in all cases (1.7%-5.9%) CONCLUSIONS: Synthetic T1WI and T2WI sequences may complement or replace conventional images in neonatal imaging, and robust synthetic volumetric results are accessible from a clinical workstation in less than 1 minute. Via the above-described methods, volume assessments could be routinely used in daily clinical practice.

Localization of filamin in smooth muscle.

The distribution of contractile and cytoskeletal proteins in smooth muscle has been mapped by immunocytochemical methods, with special reference to the localization of the actin-binding protein, filamin. Immunolabeling of ultrathin sections of polyvinylalcohol-embedded smooth muscle distinguished two domains in the smooth muscle cell: (a) actomyosin domains, made up of continuous longitudinal arrays of actin and myosin filaments, and (b) longitudinal, fibrillar, intermediate filament domains, free of myosin but containing actin and alpha-actinin-rich dense bodies. Filamin was found to be localized specifically in the latter intermediate filament-actin domains, but was excluded from the core of the dense bodies. Filamin was also localized close to the cell border at the inner surface of the plasmalemma-associated plaques. In isolated cells the surface filamin label showed a rib-like distribution similar to that displayed by vinculin. It is speculated that the two domains distinguished in these studies may reflect the existence of two functionally distinct systems: an actomyosin system required for contraction and an intermediate filament-actin system, with associated gelation proteins, that is responsible, at least in part, for the slow relaxation and tone peculiar to smooth muscle.

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus.

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.

Immunoelectron microscopic localization of the 210,000-mol wt microtubule-associated protein in cultured cells of primates.

Results from ultrastructural immunocytochemistry on glutaraldehyde-fixed cells confirmed and extended findings previously obtained with immunofluorescence. A microtubule-associated protein (MAP) of 210,000 molecular weight was shown to be specifically associated with all cytoplasmic and mitotic microtubules along their entire length in primate cells. Specific labeling with the anti-MAP antibody could not be detected on any other subcellular structures, notably the centrosomes, kinetochores, microfilaments, and intermediate filaments. Treatment with the microtubule-disrupting drug, nocodazole, induced diffusion of the MAP throughout the cytoplasm. During repolymerization of microtubules following disassembly by nocodazole, the association of the MAP with the microtubules was intermediate and complete. When cells were treated with vinblastine, the tubulin paracrystals formed were heavily stained by the antibody. Neither sodium azide nor taxol affected the association of the MAP with microtubules.

The molecular organization of myosin in stress fibers of cultured cells.

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.