Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes.

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with ß-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.

Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface.

The ER-mitochondrial junction provides a local calcium signaling domain that is critical for both matching energy production with demand and the control of apoptosis. Here, we visualize ER-mitochondrial contact sites and monitor the localized [Ca(2+)] changes ([Ca(2+)](ER-mt)) using drug-inducible fluorescent interorganelle linkers. We show that all mitochondria have contacts with the ER, but plasma membrane (PM)-mitochondrial contacts are less frequent because of interleaving ER stacks in both RBL-2H3 and H9c2 cells. Single mitochondria display discrete patches of ER contacts and show heterogeneity in the ER-mitochondrial Ca(2+) transfer. Pericam-tagged linkers revealed IP(3)-induced [Ca(2+)](ER-mt) signals that exceeded 9 microM and endured buffering bulk cytoplasmic [Ca(2+)] increases. Altering linker length to modify the space available for the Ca(2+) transfer machinery had a biphasic effect on [Ca(2+)](ER-mt) signals. These studies provide direct evidence for the existence of high-Ca(2+) microdomains between the ER and mitochondria and suggest an optimal gap width for efficient Ca(2+) transfer.

Defining the morphology and mechanism of the hemoglobin transport pathway in Plasmodium falciparum-infected erythrocytes.

Hemoglobin degradation during the asexual cycle of Plasmodium falciparum is an obligate process for parasite development and survival. It is established that hemoglobin is transported from the host erythrocyte to the parasite digestive vacuole (DV), but this biological process is not well characterized. Three-dimensional reconstructions made from serial thin-section electron micrographs of untreated, trophozoite-stage P. falciparum-infected erythrocytes (IRBC) or IRBC treated with different pharmacological agents provide new insight into the organization and regulation of the hemoglobin transport pathway. Hemoglobin internalization commences with the formation of cytostomes from localized, electron-dense collars at the interface of the parasite plasma and parasitophorous vacuolar membranes. The cytostomal collar does not function as a site of vesicle fission but rather serves to stabilize the maturing cytostome. We provide the first evidence that hemoglobin transport to the DV uses an actin-myosin motor system. Short-lived, hemoglobin-filled vesicles form from the distal end of the cytostomes through actin and dynamin-mediated processes. Results obtained with IRBC treated with N-ethylmaleimide (NEM) suggest that fusion of hemoglobin-containing vesicles with the DV may involve a soluble NEM-sensitive factor attachment protein receptor-dependent mechanism. In this report, we identify new key components of the hemoglobin transport pathway and provide a detailed characterization of its morphological organization and regulation.

Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk.

RATIONALE: Mitochondrial Ca(2+) uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca(2+) uptake despite low Ca(2+) affinity of the mitochondrial Ca(2+) uniporter. However, the existence of such mitochondrial Ca(2+) microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial outer membrane fusion, whereas Mfn2 but not Mfn1 tethers endoplasmic reticulum to mitochondria in noncardiac cells. OBJECTIVE: To elucidate roles for Mfn1 and 2 in SR-mitochondrial tethering, Ca(2+) signaling, and bioenergetic regulation in cardiac myocytes. METHODS AND RESULTS: Fruit fly heart tubes deficient of the Drosophila Mfn ortholog MARF had increased contraction-associated and caffeine-sensitive Ca(2+) release, suggesting a role for Mfn in SR Ca(2+) handling. Whereas cardiac-specific Mfn1 ablation had no effects on murine heart function or Ca(2+) cycling, Mfn2 deficiency decreased cardiomyocyte SR-mitochondrial contact length by 30% and reduced the content of SR-associated proteins in mitochondria-associated membranes. This was associated with decreased mitochondrial Ca(2+) uptake (despite unchanged mitochondrial membrane potential) but increased steady-state and caffeine-induced SR Ca(2+) release. Accordingly, Ca(2+)-induced stimulation of Krebs cycle dehydrogenases during ß-adrenergic stimulation was hampered in Mfn2-KO but not Mfn1-KO myocytes, evidenced by oxidation of the redox states of NAD(P)H/NAD(P)(+) and FADH(2)/FAD. CONCLUSIONS: Physical tethering of SR and mitochondria via Mfn2 is essential for normal interorganelle Ca(2+) signaling in the myocardium, consistent with a requirement for SR-mitochondrial Ca(2+) signaling through microdomains in the cardiomyocyte bioenergetic feedback response to physiological stress.

Perinuclear damage from nuclear envelope deterioration elicits stress responses that contribute to LMNA cardiomyopathy.

Mutations in the LMNA gene encoding lamins A/C cause an array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the perturbations emanating from LMNA mutations, an integrative understanding of the pathogenesis underlying cardiac dysfunction remains elusive. Using a novel conditional deletion model capable of translatome profiling, we observed that cardiomyocyte-specific Lmna deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Before cardiac dysfunction, Lmna-deleted cardiomyocytes displayed nuclear abnormalities, Golgi dilation/fragmentation, and CREB3-mediated stress activation. Translatome profiling identified MED25 activation, a transcriptional cofactor that regulates Golgi stress. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the Golgi. Systemic administration of modulators of autophagy or ER stress significantly delayed cardiac dysfunction and prolonged survival. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the LMNA cardiomyopathy development.

Perinuclear damage from nuclear envelope deterioration elicits stress responses that contribute to LMNA cardiomyopathy.

Mutations in the LMNA gene encoding nuclear lamins A/C cause a diverse array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the molecular perturbations emanating from LMNA mutations, an integrative understanding of the pathogenesis leading to cardiac dysfunction remains elusive. Using a novel cell-type specific Lmna deletion mouse model capable of translatome profiling, we found that cardiomyocyte-specific Lmna deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Prior to the onset of cardiac dysfunction, lamin A/C-depleted cardiomyocytes displayed nuclear envelope deterioration, golgi dilation/fragmentation, and CREB3-mediated golgi stress activation. Translatome profiling identified upregulation of Med25, a transcriptional co-factor that can selectively dampen UPR axes. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the golgi or inducing nuclear damage by increased matrix stiffness. Systemic administration of pharmacological modulators of autophagy or ER stress significantly improved the cardiac function. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the development of LMNA cardiomyopathy. Teaser: Interplay of stress responses underlying the development of LMNA cardiomyopathy.

Alignment of sarcoplasmic reticulum-mitochondrial junctions with mitochondrial contact points.

Propagation of ryanodine receptor (RyR2)-derived Ca(2+) signals to the mitochondrial matrix supports oxidative ATP production or facilitates mitochondrial apoptosis in cardiac muscle. Ca(2+) transfer likely occurs locally at focal associations of the sarcoplasmic reticulum (SR) and mitochondria, which are secured by tethers. The outer mitochondrial membrane and inner mitochondrial membrane (OMM and IMM, respectively) also form tight focal contacts (contact points) that are enriched in voltage-dependent anion channels, the gates of OMM for Ca(2+). Contact points could offer the shortest Ca(2+) transfer route to the matrix; however, their alignment with the SR-OMM associations remains unclear. Here, in rat heart we have studied the distribution of mitochondria-associated SR in submitochondrial membrane fractions and evaluated the colocalization of SR-OMM associations with contact points using transmission electron microscopy. In a sucrose gradient designed for OMM purification, biochemical assays revealed lighter fractions enriched in OMM only and heavier fractions containing OMM, IMM, and SR markers. Pure OMM fractions were enriched in mitofusin 2, an ~80 kDa mitochondrial fusion protein and SR-mitochondrial tether candidate, whereas in fractions of OMM + IMM + SR, a lighter (~50 kDa) band detected by antibodies raised against the NH(2) terminus of mitofusin 2 was dominating. Transmission electron microscopy revealed mandatory presence of contact points at the junctional SR-mitochondrial interface versus a random presence along matching SR-free OMM segments. For each SR-mitochondrial junction at least one tether was attached to contact points. These data establish the contact points as anchorage sites for the SR-mitochondrial physical coupling. Close coupling of the SR, OMM, and IMM is likely to provide a favorable spatial arrangement for local ryanodine receptor-mitochondrial Ca(2+) signaling.

The longin domain regulates the steady-state dynamics of Sec22 in Plasmodium falciparum.

The specificity of vesicle-mediated transport is largely regulated by the membrane-specific distribution of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. However, the signals and machineries involved in SNARE protein targeting to the respective intracellular locations are not fully understood. We have identified a Sec22 ortholog in Plasmodium falciparum (PfSec22) that contains an atypical insertion of the Plasmodium export element within the N-terminal longin domain. This Sec22 protein partially associates with membrane structures in the parasitized erythrocytes when expressed under the control of the endogenous promoter element. Our studies indicate that the atypical longin domain contains signals that are required for both endoplasmic reticulum (ER)/Golgi apparatus recycling of PfSec22 and partial export beyond the ER/Golgi apparatus interface. ER exit of PfSec22 is regulated by motifs within the alpha3 segment of the longin domain, whereas the recycling and export signals require residues within the N-terminal hydrophobic segment. Our data suggest that the longin domain of PfSec22 exhibits major differences from the yeast and mammalian orthologs, perhaps indicative of a novel mechanism for Sec22 trafficking in malaria parasites.

Early results of minimally invasive, reverse-oblique, distal metaphyseal metatarsal osteotomy (R-DMMO) for arthritis of the lesser tarsometatarsal joints - A retrospective case series.

BACKGROUND: Arthrodesis is the gold standard for operative management of osteoarthritis of the lesser tarsometatarsal joints (TMTJs) but is not without complications. Our early results of a minimally invasive alternative treatment - the reverse-oblique distal metaphyseal metatarsal osteotomy (R-DMMO) are described. METHODS: This is a single-centre, single-surgeon, retrospective series of patients with isolated, symptomatic lesser TMTJ arthrosis who underwent R-DMMO. RESULTS: Sixteen feet in 15 patients were included. The mean age was 64.7±9.7 years and mean duration of follow-up was 109.4±27.4 weeks. There were no non-unions, infections or wound complications. Two patients developed transfer symptoms to their first metatarsal, one of these patients improved after three months. There was one delayed union which united at 12 months. Two patients developed recurrence of symptoms but felt that they were still improved compared to preoperatively and no patient has required arthrodesis thus far. The mean preoperative VAS was 8.3±1.3 and the mean postoperative VAS was 2.4±2.2 (P<0.001). The mean postoperative MOxFQ-Walking was 25.2±25.6, MOxFQ-Pain was 24.8±20.5, MOxFQ-Social was 18.4±19.1, and MOxFQ-Index was 23.4±20.6. Eight patients were 'very satisfied' and seven were 'satisfied' with the procedure. CONCLUSIONS: R-DMMO is a minimally invasive and safe procedure for lesser TMTJ arthrosis which can produce good results and prevent, or at least delay, the need for arthrodesis without compromising future operative options. Good to excellent outcomes have been shown with few significant complications in the short term in selected patients.

Matrix-Induced Autologous Chondrocyte Implantation (MACI) Grafting for Osteochondral Lesions of the Talus.

BACKGROUND: Matrix-induced autologous chondrocyte implantation (MACI) is an established treatment method for larger joints and has shown promising results in the ankle as well. We present a series of patients after ankle MACI with long-term follow-up of clinical and radiological outcomes. METHODS: We present the follow-up of 15 patients who underwent MACI grafting from August 2003 to February 2006. The mean follow-up was 12.9 years. Clinical evaluations were conducted using the American Orthopaedic Foot & Ankle Society (AOFAS), Foot and Ankle Activity Measurement (FAAM), and visual analog scale (VAS) scoring systems and the magnetic resonance observation of cartilage repair tissue (MOCART) scoring system for radiological evaluation. RESULTS: The mean size of the talar osteochondral defects was 204 mm2. We found a significant improvement in mean AOFAS score from 60 preoperatively to a mean of 84 at 12 years postoperatively. The 12-year FAAM score for Activities of Daily Living was 89% (range, 62%-99%). The mean 12-year MOCART score was 65 points (range, 30-100 points) with significant agreement between assessors (P < .001). However, the MOCART scores did not correlate with the FAAM scores (P = .86). CONCLUSION: Considering our long-term follow-up, we believe MACI is a reliable treatment method for talar osteochondral defects providing lasting pain relief and satisfying clinical results. However, with an equivalent outcome, but at higher costs, and the requirement for 2 operative procedures, the results do not seem to be superior to other established methods. The clinical utility of the MOCART score requires further scrutiny since we were not able to show any correlation between the score and clinical outcome. LEVEL OF EVIDENCE: Level IV, case series.

Matrix-Induced Autologous Chondrocyte Implantation (MACI) grafting for osteochondral lesions of the talus.

BACKGROUND: Articular cartilage is limited in its ability to repair itself. Matrix-induced Autologous Chondrocyte Implantation (MACI) is an established treatment method for such articular cartilage defects in the knee. Recently the technique has been used in the ankle. We present a series of patients treated with MACI for osteochondral defects of the ankle, and assess the functional and clinical results. MATERIALS AND METHODS: From August 2003 to February 2006, 20 patients underwent MACI grafting for osteochondral defects in the ankle. Age ranged from 19 to 61 (mean, 36) years. Mean followup was 21.1 months. Clinical and functional evaluations were conducted using the AOFAS scoring system. RESULTS: The mean size was 233 mm(2). There was a significant improvement in mean AOFAS score from 60 (range, 25 to 87) to 87 (range, 41 to 100) (p < 0.0001). Overall improvement in pain scores was also significant (p < 0.0001). All osteotomies healed. Four patients required hardware removal and two underwent arthroscopic debridement for anterior impingement. There were two failures which are awaiting subsequent procedures. CONCLUSION: We believe MACI is a reliable treatment method for talar osteochondral defects. The method usually requires an intra-articular osteotomy, although this proved to be a reasonably simple aspect of the procedure for the treatment of cartilage defects of the talus.

Distal tibiofibular syndesmosis reconstruction using a free hamstring autograft.

BACKGROUND: Chronic instability of the distal tibiofibular syndesmosis and its treatment is infrequently described in the literature. We report our results for treating this condition with a reconstruction based on restoration of near normal anatomy with a hamstring autograft. MATERIALS AND METHODS: Eight patients, who had chronic distal tibiofibular syndesmosis instability diagnosed arthroscopically underwent reconstruction of the distal anterior tibiofibular ligament and the transverse interosseus ligament using a free semi-tendinosis hamstring autograft. A radiographic and clinical review of the patients was performed. The average followup was 39 (range, 9 to 86) months. RESULTS: The postoperative visual analogue pain score was 19 out of 100 compared to 73 out of 100 preoperatively. The postoperative mean AOFAS score was 85.4 (range, 49 to 100), the SF-36 score was 81 (range, 56 to 92) and the Maryland foot score was 89.3 (range, 63 to 100). CONCLUSION: Chronic instability of the distal tibiofibular syndesmosis is an infrequent problem but disabling when it occurs. Our results show an improved VAS score for pain, improved swelling and instability symptoms as well as SF-36, AOFAS and Maryland scores. We would recommend this method to reconstruct the anterior tibiofibular ligament and the transverse interosseus ligament which has failed to respond to debridement alone.

MR imaging and ultrasound of metatarsalgia--the lesser metatarsals.

Metatarsalgia is a common problem for many in the community. The condition includes many different entities, such as interdigital neuroma, synovitis or metatarsophalangeal joint instability, Freiberg infarction, stress fractures, and systemic disorders. Many patients presenting with metatarsalgia have a combination of diagnostic abnormalities. The key is to establish the principal pathology and from there construct an appropriate treatment regimen.

Nomogram considerations with the Technolas 217A for treatment of myopia.

PURPOSE: To determine whether surgeon-specific nomogram adjustments are useful when using the Technolas 217A excimer laser for treating myopia and myopic astigmatism. METHODS: We conducted a prospective evaluation of 216 consecutive eyes with 6 months follow-up after treatment of myopia or myopic astigmatism with the Technolas 217A laser. Attempted vs. achieved change in refraction was analyzed with a statistical analysis software program. Factors such as age, corneal thickness (pachymetry), preoperative spherical equivalent refraction, preoperative cylinder, and optical zone were studied to evaluate their role in predicting refractive outcome at 6 months after LASIK. RESULTS: The mean value of attempted spherical equivalent refraction was -5.32 +/- 2.72 D. The mean achieved refractive correction at 6 months was -5.55 +/- 2.78 D, with a mean spherical equivalent of 0.13 +/- 0.54 D. The percent achieved effect at 1 month was 105%, and at 6 months, 103%. Preoperative spherical equivalent refraction and optical zone size were strong predictors of 6-month LASIK outcome. There was a 9% difference in the percent achieved effect between a 4 and 7-mm optical zone. There was no correlation between age, preoperative cylinder, or surgeon and 6-month outcome. CONCLUSIONS: Surgeons using the planoscan software on the Technolas 217A may experience a small initial overcorrection. There may be a benefit to reducing the treatment given with larger optical zones and smaller corrections.

Epi-LASEK for the correction of myopia and myopic astigmatism.

PURPOSE: To analyze patient results after laser-assisted subepithelial keratectomy (epi-LASEK) for myopia and myopic astigmatism. SETTING: Private practice setting, Columbus, Ohio, USA. METHODS: Three hundred forty-three eyes of 188 patients with myopia or myopic astigmatism were prospectively evaluated after having epi-LASEK by a single surgeon using the VISX Star S2 excimer laser. Uncorrected visual acuity (UCVA), manifest refraction, postoperative pain, time to epithelial healing, and postoperative haze were recorded. The patients were followed for up to 6 months. RESULTS: The mean preoperative sphere and cylinder were -5.42 diopters (D) +/- 2.62 (SD) (range -1.0 to -14.0 D) and 0.87 +/- 0.75 D (range 0 to 4.75 D), respectively. At 1 week, the mean UCVA was 20/30. At 6 months, it was 20/40 or better in 98% of patients. At 3 months, 78% and 92% of patients were within +/-0.5 D and +/-1.0 D, respectively, of the intended correction. This improved to 85% and 94%, respectively, at 6 months. The mean time to epithelial healing was 4.76 days (range 3 to 9 days). Most patients (87%) reported no postoperative pain. In the first 3 months, haze was noted in 1.6% of patients. CONCLUSIONS: Epi-LASEK appeared to be a safe and effective treatment for the correction of myopia and myopic astigmatism. Most patients achieved postoperative visual acuities comparable to those with laser in situ keratomileusis and photorefractive keratectomy. There was a low incidence of haze and pain postoperatively.

A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum.

The current model for hemoglobin ingestion and transport by intraerythrocytic Plasmodium falciparum malaria parasites shares similarities with endocytosis. However, the model is largely hypothetical, and the mechanisms responsible for the ingestion and transport of host cell hemoglobin to the lysosome-like food vacuole (FV) of the parasite are poorly understood. Because actin dynamics play key roles in vesicle formation and transport in endocytosis, we used the actin-perturbing agents jasplakinolide and cytochalasin D to investigate the role of parasite actin in hemoglobin ingestion and transport to the FV. In addition, we tested the current hemoglobin trafficking model through extensive analysis of serial thin sections of parasitized erythrocytes (PE) by electron microscopy. We find that actin dynamics play multiple, important roles in the hemoglobin transport pathway, and that hemoglobin delivery to the FV via the cytostomes might be required for parasite survival. Evidence is provided for a new model, in which hemoglobin transport to the FV occurs by a vesicle-independent process.

Sonographic and MRI evaluation of the plantar plate: A prospective study.

The purpose of this study was to establish the accuracy of ultrasound in the examination of the plantar plate by comparing it with MRI, or if available, surgical findings. The lesser metatarsophalangeal joint plantar plates of 40 symptomatic and 40 asymptomatic feet (160 asymptomatic and 160 symptomatic plantar plates) were examined with ultrasound and MRI. Patients treated with surgery were chosen on a clinical basis and provided surgical correlation for the imaging techniques. Symptomatic patients with metatatarsalgia and suspected metatarsophalangeal joint instability were referred by an orthopedic foot specialist; asymptomatic feet were obtained either through examination of the contralateral foot of the symptomatic patients or volunteers. Ultrasound detected 75/160 and 139/160 plantar plates torn in the asymptomatic and symptomatic groups, respectively. MRI detected 56/160 and 142/160 tears in the symptomatic and asymptomatic groups, respectively. The sensitivity of MRI and ultrasound with surgical correlation was calculated to be 87 and 96%, respectively, with poor specificity. Ultrasound correlates moderately with MRI in the evaluation of the plantar plate. Surgical correlations, although limited (n = 10), indicate ultrasound is superior to MRI with more accurate detection of tears.

The course of etoposide-induced apoptosis in Jurkat cells lacking p53 and Bax.

Jurkat T-lymphocytes lack p53 and Bax but contain p73 and Bid and are killed by etoposide (ETO). With ETO c-abl is phosphorylated and phosphorylated p73 increased. Translocation of full-length Bid to mitochondria follows, with induction of the mitochondrial permeability transition (MPT) and release of cytochrome c into the cytosol. Pronounced swelling of mitochondria was evident ultrastructurally, and the MPT inhibitor cyclosporin A prevented the release of cytochrome c. Overexpression of Bcl-2 prevented the translocation of Bid, the release of cytochrome c, and cell death. The pan-caspase inhibitor ZVAD-FMK prevented the cell killing, but not the initial release of cytochrome c. An accumulation of tBid occurred at later times in association with Bid degradation. A sequence is proposed that couples DNA damage to Bid translocation via activation of c-abl and p73. Bid translocation induces the MPT, the event that causes release of cytochrome c, activation of caspases, and cell death.

Sonography of plantar plates in cadavers: correlation with MRI and histology.

OBJECTIVE: The purpose of our study was to describe the sonographic appearance of the lesser metatarsal plantar plates in cadavers and to correlate these findings with MRI and histology. MATERIALS AND METHODS: Six soft-embalmed cadaveric feet (74-92 years old; two male, one female) were imaged with sonography and MRI. Tear dimensions of the plantar plate were recorded in the long and short axes. Orthopedic surgeons directly inspected the plantar plates before removing samples for histologic correlation. One young fresh cadaver was imaged with sonography before histologic assessment. RESULTS: The normal plantar plate appearance on sonography was a slightly echoic, homogeneous, curved structure. At direct inspection, a tear was present in 23 (96%) of 24 of the lesser plantar plates in the soft-embalmed feet. This direct inspection correlated with sonography detecting 23 tears correctly and MRI, 22 tears. Both sonography and MRI falsely reported one tear, but MRI also failed to detect one tear. Histologically, the abnormal plantar plate showed loss of the normal dense regular tissue and replacement with vessels, hydropic tissue, and a mixture of loose connective tissue and dense irregular connective tissue. CONCLUSION: Sonography, being noninvasive, shows promise as an imaging tool of the plantar plate. With ongoing research in this area we hope to determine the reliability and significance of such a technique in the evaluation of the plantar plate.

Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains.

The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or "rafts" have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-beta-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.