PET imaging demonstrates histone deacetylase target engagement and clarifies brain penetrance of known and novel small molecule inhibitors in rat.

Histone deacetylase (HDAC) enzymes have been demonstrated as critical components in maintaining chromatin homeostasis, CNS development, and normal brain function. Evidence in mouse models links HDAC expression to learning, memory, and mood-related behaviors; small molecule HDAC inhibitor tool compounds have been used to demonstrate the importance of specific HDAC subtypes in modulating CNS-disease-related behaviors in rodents. So far, no direct evidence exists to understand the quantitative changes in HDAC target engagement that are necessary to alter biochemistry and behavior in a living animal. Understanding the relationship between target engagement and in vivo effect is essential in refining new ways to alleviate disease. We describe here, using positron emission tomography (PET) imaging of rat brain, the in vivo target engagement of a subset of class I/IIb HDAC enzymes implicated in CNS-disease (HDAC subtypes 1, 2, 3, and 6). We found marked differences in the brain penetrance of tool compounds from the hydroxamate and benzamide HDAC inhibitor classes and resolved a novel, highly brain penetrant benzamide, CN147, chronic treatment with which resulted in an antidepressant-like effect in a rat behavioral test. Our work highlights a new translational path for understanding the molecular and behavioral consequences of HDAC target engagement.

Epigenetic mechanisms in mood disorders: targeting neuroplasticity.

Developing novel therapeutics and diagnostic tools based upon an understanding of neuroplasticity is critical in order to improve the treatment and ultimately the prevention of a broad range of nervous system disorders. In the case of mood disorders, such as major depressive disorder (MDD) and bipolar disorder (BPD), where diagnoses are based solely on nosology rather than pathophysiology, there exists a clear unmet medical need to advance our understanding of the underlying molecular mechanisms and to develop fundamentally new mechanism experimental medicines with improved efficacy. In this context, recent preclinical molecular, cellular, and behavioral findings have begun to reveal the importance of epigenetic mechanisms that alter chromatin structure and dynamically regulate patterns of gene expression that may play a critical role in the pathophysiology of mood disorders. Here, we will review recent advances involving the use of animal models in combination with genetic and pharmacological probes to dissect the underlying molecular mechanisms and neurobiological consequence of targeting this chromatin-mediated neuroplasticity. We discuss evidence for the direct and indirect effects of mood stabilizers, antidepressants, and antipsychotics, among their many other effects, on chromatin-modifying enzymes and on the epigenetic state of defined genomic loci, in defined cell types and in specific regions of the brain. These data, as well as findings from patient-derived tissue, have also begun to reveal alterations of epigenetic mechanisms in the pathophysiology and treatment of mood disorders. We summarize growing evidence supporting the notion that selectively targeting chromatin-modifying complexes, including those containing histone deacetylases (HDACs), provides a means to reversibly alter the acetylation state of neuronal chromatin and beneficially impact neuronal activity-regulated gene transcription and mood-related behaviors. Looking beyond current knowledge, we discuss how high-resolution, whole-genome methodologies, such as RNA-sequencing (RNA-Seq) for transcriptome analysis and chromatin immunoprecipitation-sequencing (ChIP-Seq) for analyzing genome-wide occupancy of chromatin-associated factors, are beginning to provide an unprecedented view of both specific genomic loci as well as global properties of chromatin in the nervous system. These methodologies when applied to the characterization of model systems, including those of patient-derived induced pluripotent cell (iPSC) and induced neurons (iNs), will greatly shape our understanding of epigenetic mechanisms and the impact of genetic variation on the regulatory regions of the human genome that can affect neuroplasticity. Finally, we point out critical unanswered questions and areas where additional data are needed in order to better understand the potential to target mechanisms of chromatin-mediated neuroplasticity for novel treatments of mood and other psychiatric disorders.

Visualizing epigenetics: current advances and advantages in HDAC PET imaging techniques.

Abnormal gene regulation as a consequence of flawed epigenetic mechanisms may be central to the initiation and persistence of many human diseases. However, the association of epigenetic dysfunction with disease and the development of therapeutic agents for treatment are slow. Developing new methodologies used to visualize chromatin-modifying enzymes and their function in the human brain would be valuable for the diagnosis of brain disorders and drug discovery. We provide an overview of current invasive and noninvasive techniques for measuring expression and functions of chromatin-modifying enzymes in the brain, emphasizing tools applicable to histone deacetylase (HDAC) enzymes as a leading example. The majority of current techniques are invasive and difficult to translate to what is happening within a human brain in vivo. However, recent progress in molecular imaging provides new, noninvasive ways to visualize epigenetics in the human brain. Neuroimaging tool development presents a unique set of challenges in order to identify and validate CNS radiotracers for HDACs and other histone-modifying enzymes. We summarize advances in the effort to image HDACs and HDAC inhibitory effects in the brain using positron emission tomography (PET) and highlight generalizable techniques that can be adapted to investigate other specific components of epigenetic machinery. Translational tools like neuroimaging by PET and magnetic resonance imaging provide the best way to link our current understanding of epigenetic changes with in vivo function in normal and diseased brains. These tools will be a critical addition to ex vivo methods to evaluate - and intervene - in CNS dysfunction.

Chemical defense of an Asian snake reflects local availability of toxic prey and hatchling diet.

Species that sequester toxins from prey for their own defense against predators may exhibit population-level variation in their chemical arsenal that reflects the availability of chemically defended prey in their habitat. Rhabdophis tigrinus is an Asian snake that possesses defensive glands in the skin of its neck ('nuchal glands'), which typically contain toxic bufadienolide steroids that the snakes sequester from consumed toads. In this study, we compared the chemistry of the nuchal gland fluid of R. tigrinus from toad-rich and toad-free islands in Japan and determined the effect of diet on the nuchal gland constituents. Our findings demonstrate that captive-hatched juveniles from toad-rich Ishima Island that had not been fed toads possess defensive bufadienolides in their nuchal glands, presumably due to maternal provisioning of these sequestered compounds. Wild-caught juveniles from Ishima possess large quantities of bufadienolides, which could result from a combination of maternal provisioning and sequestration of these defensive compounds from consumed toads. Interestingly, juvenile females from Ishima possess larger quantities of bufadienolides than do juvenile males, whereas a small sample of field-collected snakes suggests that adult males contain larger quantities of bufadienolides than do adult females. Captive-born hatchlings from Kinkasan Island lack bufadienolides in their nuchal glands, reflecting the absence of toads on that island, but they can sequester bufadienolides by feeding on toads (Bufo japonicus) in captivity. The presence of large quantities of bufadienolides in the nuchal glands of R. tigrinus from Ishima may reduce the risk of predation by providing an effective chemical defense, whereas snakes on Kinkasan may experience increased predation due to the lack of defensive compounds in their nuchal glands.

Amorfrutin B is an efficient natural peroxisome proliferator-activated receptor gamma (PPARγ) agonist with potent glucose-lowering properties.

AIMS/HYPOTHESIS: The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is an important gene regulator in glucose and lipid metabolism. Unfortunately, PPARγ-activating drugs of the thiazolidinedione class provoke adverse side effects. As recently shown, amorfrutin A1 is a natural glucose-lowering compound that selectively modulates PPARγ. In this study we aimed to characterise, in vitro, a large spectrum of the amorfrutins and similar molecules, which we isolated from various plants. We further studied in vivo the glucose-lowering effects of the so far undescribed amorfrutin B, which featured the most striking PPARγ-binding and pharmacological properties of this family of plant metabolites. METHODS: Amorfrutins were investigated in vitro by binding and cofactor recruitment assays and by transcriptional activation assays in primary human adipocytes and murine preosteoblasts, as well as in vivo using insulin-resistant high-fat-diet-fed C57BL/6 mice treated for 27 days with 100 mg kg(-1) day(-1) amorfrutin B. RESULTS: Amorfrutin B showed low nanomolar binding affinity to PPARγ, and micromolar binding to the isotypes PPARα and PPARß/δ. Amorfrutin B selectively modulated PPARγ activity at low nanomolar concentrations. In insulin-resistant mice, amorfrutin B considerably improved insulin sensitivity, glucose tolerance and blood lipid variables after several days of treatment. Amorfrutin B treatment did not induce weight gain and furthermore showed liver-protecting properties. Additionally, amorfrutins had no adverse effects on osteoblastogenesis and fluid retention. CONCLUSIONS/INTERPRETATION: The application of plant-derived amorfrutins or synthetic analogues thereof constitutes a promising approach to prevent or treat complex metabolic diseases such as insulin resistance or type 2 diabetes.

Type A aortic dissection in pregnancy.

Type A aortic dissection is a life-threatening event to both mother and baby, and accounted for 14% of maternal cardiac deaths in the 2006-2008 UK Confidential Enquiries into Maternal Deaths. Difficulty exists in the diagnosis of this rare but potentially curable condition, the mortality of which increases with delay in diagnosis. We present a case of acute type A aortic dissection in a previously well multiparous woman, treated successfully by aortic root repair immediately following caesarean section. The acute presentation of aortic dissection and diagnostic clues that may have expedited the diagnosis are discussed. A brief literature review is presented of the perioperative management of patients undergoing cardiothoracic surgery post-caesarean section and the modifications to standard techniques that are required.

Biomechanical advantages of triple-loaded suture anchors compared with double-row rotator cuff repairs.

PURPOSE: To evaluate the strength and suture-tendon interface security of various suture anchors triply and doubly loaded with ultrahigh-molecular weight polyethylene-containing sutures and to evaluate the relative effectiveness of placing these anchors in a single-row or double-row arrangement by cyclic loading and then destructive testing. METHODS: The infraspinatus muscle was reattached to the original humeral footprint by use of 1 of 5 different repair patterns in 40 bovine shoulders. Two single-row repairs and three double-row repairs were tested. High-strength sutures were used for all repairs. Five groups were studied: group 1, 2 triple-loaded screw suture anchors in a single row with simple stitches; group 2, 2 triple-loaded screw anchors in a single row with simple stitches over a fourth suture passed perpendicularly ("rip-stop" stitch); group 3, 2 medial and 2 lateral screw anchors with a single vertical mattress stitch passed from the medial anchors and 2 simple stitches passed from the lateral anchors; group 4, 2 medial double-loaded screw anchors tied in 2 mattress stitches and 2 push-in lateral anchors capturing the medial sutures in a "crisscross" spanning stitch; and group 5, 2 medial double-loaded screw anchors tied in 2 mattress stitches and 2 push-in lateral anchors creating a "suture-bridge" stitch. The specimens were cycled between 10 and 180 N at 1.0 Hz for 3,500 cycles or until failure. Endpoints were cyclic loading displacement (5 and 10 mm), total displacement, and ultimate failure load. RESULTS: A single row of triply loaded anchors was more resistant to stretching to a 5- and 10-mm gap than the double-row repairs with or without the addition of a rip-stop suture (P < .05). The addition of a rip-stop stitch made the repair more resistant to gap formation than a double row repair (P < .05). The crisscross double row created by 2 medial double-loaded suture anchors and 2 lateral push-in anchors stretched more than any other group (P < .05). CONCLUSIONS: Double-row repairs with either crossing sutures or 4 separate anchor points were more likely to fail (5- or 10-mm gap) than a single-row repair loaded with 3 simple sutures. CLINICAL RELEVANCE: The triple-loaded anchors with ultrahigh-molecular weight polyethylene-containing sutures placed in a single row were more resistant to stretching than the double-row groups.

Biomechanical testing of new meniscal repair techniques containing ultra high-molecular weight polyethylene suture.

PURPOSE: To evaluate the biomechanical characteristics of current meniscal repair techniques containing ultra high-molecular weight polyethylene (UHMWPE) suture with and without cyclic loading. METHODS: Vertical longitudinal cuts made in porcine menisci were secured with a single repair device. Noncycled and cycled (500 cycles) biomechanical tests were performed on the following groups: group 1, No. 2-0 Mersilene vertical suture (Ethicon, Somerville, NJ); group 2, No. 2-0 Orthocord vertical suture (DePuy Mitek, Westwood, MA); group 3, No. 0 Ultrabraid vertical suture (Smith & Nephew Endoscopy, Andover, MA); group 4, No. 2-0 FiberWire vertical suture (Arthrex, Naples, FL); group 5, vertically oriented mattress suture by use of an Ultra FasT-Fix device (Smith & Nephew Endoscopy) with No. 0 Ultrabraid; group 6, vertically oriented mattress suture by use of a RapidLoc A2 device (DePuy Mitek) with No. 2-0 Orthocord suture; group 7, vertically oriented stitch by use of a MaxFire device with MaxBraid PE suture (Biomet Sports Medicine, Warsaw, IN); and group 8, an obliquely oriented stitch of No. 0 UHMWPE suture inserted by use of a CrossFix device (Cayenne Medical, Scottsdale, AZ). Endpoints were failure loads, failure modes, stiffness, and cyclic displacement. RESULTS: Mean single-pull loads were calculated for Ultra FasT-Fix (121 N), FiberWire (110 N), MaxFire (130 N), Mersilene (84 N), Orthocord (124 N), RapidLoc A2 (86 N), CrossFix (77 N), and Ultrabraid (109 N). After 500 cyclic loads, the Orthocord (222 N) repair was stronger than the others: Ultra FasT-Fix (110 N), FiberWire (117 N), MaxFire (132 N), Mersilene (89 N), RapidLoc A2 (108 N), CrossFix (95 N), and Ultrabraid (126 N) (P < .05). Ultrabraid suture showed significantly more elongation over 500 cycles than the other repairs (P < .05). The principal failure mode associated with the single destructive pull (suture breakage) changed to pulling through the meniscus after cyclic loading for most devices. Knot slippage or device failure was seldom observed as the failure mode with these techniques. CONCLUSIONS: Self-adjusting, UHMWPE suture-containing meniscal repair devices (Ultra FasT-Fix, RapidLoc A2, and MaxFire) were comparable to the isolated UHMWPE-containing suture repairs on single-failure load testing. UHMWPE-containing suture repairs are stronger than braided polyester suture repairs, but pure UHMWPE suture (Ultrabraid) elongated more during cycling. Orthocord suture is significantly stronger than the other meniscal repair techniques after cyclic loading (P < .05). CLINICAL RELEVANCE: Meniscal repair techniques using UHMWPE containing sutures provide greater strength than earlier generations of meniscal repair techniques.

Antenatal blood patch in a pregnant woman with spontaneous intracranial hypotension.

Spontaneous intracranial hypotension is a condition that presents with postural headaches similar to those caused by accidental dural puncture. The diagnosis is based on clinical presentation, cerebrospinal fluid evaluation and magnetic resonance imaging scanning. We present a case of spontaneous intracranial hypotension with typical clinical and magnetic resonance imaging findings in a pregnant patient who was treated with an epidural blood patch. The blood patch, performed at 32 weeks of gestation, produced transient improvement in symptoms but failed to completely cure the headache, which worsened over the next few days. Symptoms resolved over the subsequent three weeks with conservative therapy.

[Massive subretinal bleeding--treat it or leave it?]. / Massive subretinale Blutung bei AMD am letzten Auge - behandeln oder nicht?

In eyes with massive subretinal haemorrhages, ambulatory vision can be achieved by pars plana vitrectomy and subretinal surgery. In patients with only one functional eye, this treatment is important to preserve the quality of life. The resulting large pigment epithelial defect limits visual rehabilitation. For the same reason, rTPA/gas injection or a macular translocation are possible but not sufficient in these cases.

FasT-Fix meniscal repair: mid-term results.

PURPOSE: The purpose of this study was to evaluate the clinical success of the FasT-Fix meniscal repair device (Smith & Nephew Endoscopy, Andover, MA) associated with an accelerated rehabilitation program. METHODS: A prospectively collected consecutive series of meniscal repairs performed with the FasT-Fix device was studied. The Lysholm, Tegner, Cincinnati, and International Knee Documentation Committee activity scores, along with the clinical examination findings and adverse events, were recorded for all patients. Associated procedures were recorded. An accelerated postoperative rehabilitation program was followed, independent of concurrent anterior cruciate ligament surgery. RESULTS: Forty-one meniscal repairs were performed, with an average follow-up of 30.7 months (range, 12 to 58 months). Twenty-nine of 41 repairs were performed in conjunction with anterior cruciate ligment reconstruction. The other repairs were in stable knees. There were 26 medial and 15 lateral meniscus repairs. Both menisci were repaired in 5 knees. Repeat arthroscopies were performed for 12 repairs and 7 (17%) were found to have failed. The preoperative and postoperative Lysholm, Tegner, Cincinnati, and International Knee Documentation Committee activity scores were 47.3 and 87.4, 3.4 and 7.2, 38.7 and 82.8, and 2.3 and 3.4, respectively. CONCLUSIONS: The FasT-Fix meniscal repair associated with an accelerated rehabilitation program resulted in clinically effective meniscal repair in 83% at the time of follow-up. Clinical outcome measures all improved. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Identification of the amino-acetonitrile derivative monepantel (AAD 1566) as a new anthelmintic drug development candidate.

Anthelmintic resistance has become a global phenomenon in gastro-intestinal nematodes of farm animals, including multi-drug resistance against the three major classes of anthelmintics. There is an urgent need for an anthelmintic with a new mode of action. The recently discovered amino-acetonitrile derivatives (AADs) offer a new class of synthetic chemicals with anthelmintic activity. The evaluation of AADs was pursued applying in vitro assays and efficacy and tolerability studies in rodents, sheep, and cattle. Amongst various suitable compounds, AAD 1566 eliminated many tested pathogenic nematode species, both at larval and adult stages, at a dose of 2.5 mg/kg bodyweight in sheep and 5.0 mg/kg bodyweight in cattle. The same doses were sufficient to cure animals infected with resistant or multi-drug-resistant nematode isolates. These findings, complemented by the good tolerability and low toxicity to mammals, suggest that AAD 1566, monepantel, would be a suitable anthelmintic drug development candidate.

[Multifocal chorioretinitis, papillitis, and recurrent optic neuritis in cat-scratch disease]. / Choriorétinite multifocale, papillite et névrite récurrente dans la maladie des griffes du chat.

To evaluate the causative factor for multifocal chorioretinitis, papillitis, and recurrent optic neuritis. A 41-year-old patient presenting multifocal choroiditis, papillitis, and recurrent optic neuritis was evaluated with funduscopy, angiography (FA), optical coherence tomography (OCT), visual evoked potentials (VEP), and numerous blood laboratory tests. FA and OCT showed multifocal pigment epithelial detachments. VEP showed typical changes for optic neuritis and papillitis. Indirect fluorescent antibody assay disclosed Bartonella henselae. Although cat-scratch disease frequently presents with optic neuritis or neuroretinitis, additional multifocal chorioretinal lesions associated with serous pigment epithelial detachments may occur. In case of recurrent episodes, a detailed laboratory work-up is mandatory to define the appropriate diagnosis and treatment.

Comparison of SIVmac239(352-382) and SIVsmmPBj41(360-390) enterotoxic synthetic peptides.

To characterize the active domain of the simian immunodeficiency virus (SIV) surface unit (SU) enterotoxin, peptides corresponding to the V3 loop of SIVmac239 (SIVmac) and SIVsmmPBj41 (SIVpbj) were synthesized and examined for enterotoxic activity, alpha-helical structure, and interaction(s) with model membranes. SIVmac and SIVpbj induced a dose-dependent diarrhea in 6-8-day-old mouse pups similar to full-length SU. The peptides mobilized [Ca(2+)](i) in HT-29 cells with distinct oscillations and elevated inositol triphosphate levels. Circular dichroism analyses showed the peptides were predominantly random coil in buffer, but increased in alpha-helical content when placed in a hydrophobic environment or with cholesterol-containing membrane vesicles that are rich in anionic phospholipids. None of the peptides underwent significant secondary structural changes in the presence of neutral vesicles indicating ionic interactions were important. These data show that the SIV SU enterotoxic domain localizes in part to the V3 loop region and interacts with anionic membrane domains on the host cell surface.

Sterol carrier protein-2: structure reveals function.

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.

Gene structure, intracellular localization, and functional roles of sterol carrier protein-2.

Since its discovery three decades ago, sterol carrier protein-2 (SCP-2) has remained a fascinating protein whose physiological function in lipid metabolism remains an enigma. Its multiple proposed functions arise from its complex gene structure, post-translational processing, intracellular localization, and ligand specificity. The SCP-2 gene has two initiation sites coding for proteins that share a common 13 kDa SCP-2 C-terminus: (1) One site codes for 58 kDa SCP-x which is partially post-translationally cleaved to 13 kDa SCP-2 and a 45 kDa protein. (2) A second site codes for 15 kDa pro-SCP-2 which is completely post-translationally cleaved to 13 kDa SCP-2. Very little is yet known regarding how the relative proportions of the two transcripts are regulated. Although all three proteins contain a C-terminal SKL peroxisomal targeting sequence, it is unclear why all three proteins are not exclusively localized in peroxisomes. However, the recent demonstration that the SCP-2 N-terminal presequence in pro-SCP-2 dramatically modulated the intracellular targeting coded by the C-terminal peroxisomal targeting sequence may account for the observation that as much as half of total SCP-2 is localized outside the peroxisome. The tertiary and secondary structure of the 13 kDa SCP-2, but not that of 15 kDa pro-SCP-2 and 58 kDa SCP-x, are now resolved. Increasing evidence suggests that the 58 kDa SCP-x and 45 kDa proteins are peroxisomal 3-ketoacyl-CoA-thiolases involved in the oxidation of branched chain fatty acids. Since 15 kDa pro-SCP-2 is post-translationally completely cleaved to 13 kDa SCP-2, relatively little attention has been focused on this protein. Finally, although the 13 kDa SCP-2 is the most studied of these proteins, because it exhibits diversity of its ligand partners (fatty acids, fatty acyl CoAs, cholesterol, phospholipids), new potential physiological function(s) are still being proposed and questions regarding potential compensation by other proteins with overlapping specificity are only beginning to be resolved.

Recent advances in membrane microdomains: rafts, caveolae, and intracellular cholesterol trafficking.

Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor/lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor/caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. Likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier protein-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomains in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of the lysosomal membrane, and/or intracellular cholesterol trafficking to select membranous organelles. Resolving these cholesterol pathways and the role of membrane cholesterol microdomains is essential to our understanding not only of processes that affect cholesterol metabolism, but also of the abnormal regulation that may lead to disease (diabetes, obesity, atherosclerosis, neutral lipid storage, Niemann-Pick C, congenital lipoid adrenal hyperplasia, etc.).

Production of a cyanogenic secretion by a thyridid caterpillar (Calindoea trifascialis, Thyrididae, Lepidoptera).

The thyridid caterpillar, Calindoea trifascialis, when disturbed, emits a defensive secretion from two sac-like glands that open dorsolaterally on the first abdominal segment. The larva has two arm-like protuberances that project outward from the body just in front of the gland openings. These "arms", which are wetted by secretion when the larva activates its glands, appear to function specifically for administration of the fluid. A primary component of the secretion in mandelonitrile, a cyanogenic compound, but the fluid also contains other potential deterrents, including benzaldehyde, benzoic acid, (E,E)-alpha-farnesene, and 3-methylbutyl-3-methylbutanoate. Tests done in the field in Vietnam, where the species is native, showed the secretion to be protective against ants.