Genetic galactocerebrosidase deficiency (globoid cell leukodystrophy, Krabbe disease) in rhesus monkeys (Macaca mulatta)

Globoid cell leukodystrophy, or Krabbe disease, is a severe disorder of the peripheral and central nervous system myelin caused by deficient galactocerebrosidase (GALC) activity. This autosomal recessive disease affects humans and animals including dogs, mice, and rhesus monkeys. Cloning of the human and animal GALC genes opened opportunities for therapeutic trials using animal models. We describe the clinical, pathologic, and biochemical features of the affected rhesus monkey. Affected monkeys had very low GALC activity and a two base pair deletion in both copies of the GALC gene. Clinical signs of tremors, hypertonia, and incoordination led to humane euthanasia by 5 months of age. At necropsy, peripheral nerves were enlarged. Microscopically, the cerebral, cerebellar, and spinal cord white matter was infiltrated with periodic acid-Schiff-positive multinucleated globoid cells, and there was a striking lack of myelin. Peripheral nerve fibers were decreased in number and separated by Alcian blue- and safranin O-positive material. Myelin sheaths were greatly diminished. Lipid analysis of brains of 12-day-old and 158-day-old affected monkeys revealed a great excess of psychosine in white matter. The rhesus monkey model will be especially useful for exploring treatment options, including prenatal bone marrow transplantation and various approaches to gene therapy.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube

The actin cytoskeleton plays a crucial role in the growth and polarity of the pollen tube. Due to inconsistencies in the conventional preservation methods, we lack a unified view of the organization of actin microfilaments, especially in the apical domain, where tip growth occurs. In an attempt to improve fixation methods, we have developed a rapid freeze-whole mount procedure, in which growing pollen tubes (primarily lily) are frozen in liquid propane at –180°C, substituted at –80°C in acetone containing glutaraldehyde, rehydrated, quenched with sodium borohydride, and probed with antibodies. Confocal microscopy reveals a distinct organization of actin in the apical domain that consists of a dense cortical fringe or collar of microfilaments starting about 1–5 m behind the extreme apex and extending basally for an additional 5–10 m. In the shank of the pollen tube, basal to the fringe, actin forms abundant longitudinal filaments that are evenly dispersed throughout the cytoplasm. We have also developed an improved ambient-temperature chemical fixation procedure, modified from a protocol based on simultaneous fixation and phalloidin staining. We removed EGTA, elevated the pH to 9, and augmented the fixative with ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS). Notably, this protocol preserves the actin cytoskeleton in a pattern similar to that produced by cryofixation. These procedures provide a reproducible way to preserve the actin cytoskeleton; employing them, we find that a cortical fringe in the apex and finely dispersed longitudinal filaments in the shank are consistent features of the actin cytoskeleton.     

The fine structure of polychaete septate junctions

Summary Epidermal septate junctions of Nereis sp. and Cirriformia sp. fixed with OsO₄ or glutaraldehyde/OsO₄ display variable structure in electron micrographs. In transverse section the septa are often indistinct and obscured by opaque material that fills the junctional cleft. Septa (spaced at 180–280 Å) are more clearly defined in slightly oblique transverse section; they exhibit an electron lucent center and appear to be linked by arms. En face views of the junction show a honeycomb pattern. Cytoplasmic faces of junctional membranes are backed with plaques opposite the septa. Lanthanum used as a tracer delineates junctional structure in negative contrast. In transverse section a chain-like lattice is present in the junctional cleft. En face views show parallel rows of pleated elements often linked by arms into honeycomb arrays. Oblique sections demonstrate that these pleated elements are continuous with the chain-like lattice seen in transverse sections. Lanthanum does not pass entirely through the junction. Lanthanum reveals that the septa have a very intricate substructure, but it is difficult to visualize the architecture that could generate the various images presented by these junctions when seen in different orientations. However, it is clear that these junctions possess some features that are diagnostic of several supposedly different types of septate junctions in invertebrates.Supported by USPHS grants NIH 5 P01 NS-07512, NIH 2701 GM-00102, and NB-00840, and by a grant from the Pomona College Research CommitteeI thank Sarah Wurzelmann, Stanley Brown, Nancy Kelly, and Gerhard Ott for excellent technical assistance. Portions of this study were carried out while I was a Postdoctoral Fellow in the Department of Anatomy, Albert Einstein College of Medicine. I dedicate this article to Berta Scharrer as a token of appreciation and affection for her guidance, encouragement, inspiration, and example of excellence     

Long-term effects of graduated compression stockings on cardiorespiratory performance

The use of graduated compression stockings (GCS) in sport has been increasing in the last years due to their potential positive effects for athletes. However, there is little evidence to support whether these types of garments actually improve cardiorespiratory performance. The aim of this study was to examine the cardiorespiratory responses of GCS during running after three weeks of regular use. Twenty recreational runners performed three tests on different days: test 1) – a 5-min maximal effort run in order to determine the participants’ maximal aerobic speed; and tests 2) and 3) – a fatigue running test of 30 minutes at 80% of their maximal aerobic speed with either GCS or PLACEBO stockings at random. Cardiorespiratory parameters (minute ventilation, heart rate, relative oxygen consumption, relative carbon dioxide production, ventilatory equivalents for oxygen and carbon dioxide, and oxygen pulse) were measured. Before each test in the laboratory, the participants trained with the randomly assigned stockings (GCS or PLACEBO) for three weeks. No significant differences between GCS and PLACEBO were found in any of the cardiorespiratory parameters. In conclusion, the present study provides evidence that running with GCS for three weeks does not influence cardiorespiratory parameters in recreational runners.     

The autumn effect: timing of physical dormancy break in seeds of two winter annual species of Geraniaceae by a stepwise process

Background and Aims

The involvement of two steps in the physical dormancy (PY)-breaking process previously has been demonstrated in seeds of Fabaceae and Convolvulaceae. Even though there is a claim for a moisture-controlled stepwise PY-breaking in some species of Geraniaceae, no study has evaluated the role of temperature in the PY-breaking process in this family. The aim of this study was to determine whether a temperature-controlled stepwise PY-breaking process occurs in seeds of the winter annuals Geranium carolinianum and G. dissectum.

Methods

Seeds of G. carolinianum and G. dissectum were stored under different temperature regimes to test the effect of storage temperature on PY-break. The role of temperature and moisture regimes in regulating PY-break was investigated by treatments simulating natural conditions. Greenhouse (non-heated) experiments on seed germination and burial experiments (outdoors) were carried out to determine the PY-breaking behaviour in the natural habitat.

Key Results

Irrespective of moisture conditions, sensitivity to the PY-breaking step in seeds of G. carolinianum was induced at temperatures ≥20 °C, and exposure to temperatures ≤20 °C made the sensitive seeds permeable. Sensitivity of seeds increased with time. In G. dissectum, PY-break occurred at temperatures ≥20 °C in a single step under constant wet or dry conditions and in two steps under alternate wet–dry conditions if seeds were initially kept wet.

Conclusions

Timing of seed germination with the onset of autumn can be explained by PY-breaking processes involving (a) two temperature-dependent steps in G. carolinianum and (b) one or two moisture-dependent step(s) along with the inability to germinate under high temperatures in G. dissectum. Geraniaceae is the third of 18 families with PY in which a two-step PY-breaking process has been demonstrated.     

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2: INDISPENSABILITY OF THE STILBENOID CORE*

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.