Preformed frozen sucrose gradients--a new laboratory aid

A useful method for preparing large numbers of virtually identical gradients of sucrose has been described. This technique involves storage of preformed gradients at ?60°C for future use after a 90 min thaw at room temperature. The method is applicable to salt-containing sucrose gradients with certain limitations, noted above.     

Basement membrane lectin binding sites are decreased in the esophageal endoderm during the arrival of presumptive muscle mesenchyme in the developing asteroid Pisaster ochraceus.

Basement membranes (BMs) of vertebrates and invertebrates have been shown to contain glycoproteins and proteoglycans, which include oligosaccharides and glycosaminoglycans. Lectin binding sites were characterized in the BM of gastrulating embryos of the starfish, Pisaster ochraceus. In early and mid-gastrulae, the fluorescein isothiocyanate (FITC)-lectin conjugates of concanavalin A (Con A) and wheat germ agglutinin (WGA) reveal the presence of mannose/glucose and glucosamine/sialic acid residues in the BM of all regions of the embryos. However, in the late gastrula embryo, an apparent reduction of these components is observed over the esophageal BM. Ultrastructural studies using the lectin-gold conjugates Con A, Limax flavus agglutinin (LFA), specific for sialic acid, and Dolichos biflorus agglutinin (DBA), specific for galactosamine, demonstrate that most mannose/glucose and galactosamine-containing residues lie in the lamina densa, whereas most sialic acid residues are located over the lamina lucida. In addition, a statistical analysis of lectin binding in the late gastrula embryo reveals that the amount of labelling with both Con A and LFA is significantly reduced in the esophageal region, suggesting that mannose/glucose and sialic acid residues are reduced in this region. These results confirm the observations of the FITC-lectin studies described above. They also confirm earlier studies that demonstrated a difference in BM morphology of the esophageal region (Crawford, '88). Mesenchyme cells, some of which arise from the forming coeloms (Crawford, '90), and which may represent a distinct population, colonize exclusively on this esophageal BM, where they later differentiate into muscle. Quantitative differences in BM glycoconjugates may act to direct the presumptive muscle cells to the region of the esophagus.     

Tentoxin-induced energy-independent adenine nucleotide exchange and ATPase activity with chloroplast coupling factor 1.

1. The effect of energy transfer inhibitors on energy-dependent exchange of tightly bound adenine nucleotides with washed, broken spinach thylakoids has been studied. Energy transfer inhibitors that inhibit the ATPase activity of soluble chloroplast coupling factor 1 (CF1) (e.g. phloridzin and tentoxin) do not inhibit energy-dependent adenine nucleotide exchange. Energy transfer inhibitors that block proton flux through the hydrophobic protein proton channel (CF0) (e.g. dicyclohexylcarbodiimide and triphenyltin chloride) also block light-dependent adenine nucleotide exchange. 2. Tentoxin, at relatively high concentrations, stimulates an energy-independent exchange of adenosine diphosphate. 3. High concentrations of tentoxin elicit a Ca2+-dependent ATPase activity with soluble CF1, but has no effect on the Ca2+-dependent ATPase activity of membrane-bound CF1. 4. The trypsin-activated, Ca2+-dependent, membrane-bound ATPase is not affected by high concentrations of tentoxin, whereas the dithiothreitol-activated, Mg2+-dependent ATPase is markedly inhibited. 5. The reconstitution of chloroplasts, partially depleted in CF1, with soluble CF1 is correlated with the loss of tentoxin-induced, Ca2+-dependent ATPase activity associated with soluble CF1.     

Yeast silencers create domains of nuclease-resistant chromatin in an SIR4-dependent manner

Previous analysis of the repression of the silent mating type loci in Saccharomyces cerevisiae has linked the mechanism of silencing to the formation of a chromatin domain at the silenced loci. In this study, a TRP1 reporter gene was used to examine changes in chromatin structure in a neutral environment. This enabled the chromatin structure organized by yeast silencers to be compared directly with changes effected by the yeast α2 repressor. It was found that silencers mediate the formation of lengthy nuclease-resistant domains on the DNA, rather than specifically positioning nucleosomes over promoter regions as the α2 repressor does. Silencing at the TRP1 reporter gene closely resembled silencing at the HMR and HML loci. Repression of the test gene was optimal when two silencers flanking the reporter gene were used, mimicking the situation at the silent loci. In addition, both repression of the reporter gene and the formation of nuclease-resistant chromatin domains was SIR4 dependent. Received: 31 October 1996; in revised form: 6 March 1997 / Accepted: 25 March 1997     

Heterochromatin protein HP1Hsβ(p25β) and its localization with centromeres in mitosis

Some autoimmune sera containing anticentromere autoantibodies also recognize a doublet of Mr 23 000 (p23) and 25 000 (p25) in addition to CENP (centromere protein)-A (Mr 19 000), -B (Mr 80 000), and -C (Mr 140 000). A p25 antigen (HP1^Hsα) has been shown to be a human homolog of Drosophila HP1 (heterochromatin protein 1). We have isolated a cDNA clone encoding another form of p25 (HP1^Hsβor p25β) from a λZap HepG2 library using human autoimmune serum. The deduced amino acid sequence of the clone contained a conserved chromodomain (chromatin modifier domain) in the N-terminal region and a heterochromatin binding domain in the C-terminal region. In immunofluorescence experiments, only affinity purified antibodies reactive with the C-terminal (amino acids 70–185) domain showed nucleoplasmic and heterochromatin staining, whereas N-terminal (amino acids 1–115) specific antibodies were nonreactive. In metaphase chromosome spreads, the C-terminal domain antibody was also localized to the centromeric regions of chromosomes. Association with centromeres was most prominent at anaphase and changed to a more generalized association with whole chromosomes in telophase. The cooccurrence of autoantibodies to centromere proteins and HP1 in certain autoimmune diseases might be a reflection of coordinated immune responses to these closely associated sets of proteins. Received: 8 August 1996; in revised form: 4 December 1996 / Accepted: 17 December 1996     

Non-seasonal clade-specificity and subclade microvariation in symbiotic dinoflagellates (Symbiodinium spp.) in Zoanthus sansibaricus (Anthozoa: Hexacorallia) at Kagoshima Bay, Japan

While much work has investigated the genetic diversity of symbiotic dinoflagellate genus Symbiodinium Freudenthal in cnidarians, investigations into such diversity over temporal scales (seasonal and/or annual) remain scarce. Here, we have sequenced the internal transcribed spacer of ribosomal DNA (ITS‐rDNA) of Symbiodinium from samples of designated Zoanthus sansibaricus Carlgren (Anthozoa: Hexacorallia) colonies collected for 12 months (August 2004–July 2005) at a high latitude non‐reefal coral community at Sakurajima, Kagoshima Bay, Japan (31°35′N, 130°35′E). Our results show that despite large ocean temperature changes (15.0–29.0°C) throughout the one‐year experimental period, Z. sansibaricus colonies contained only clade C Symbiodinium from many different subclade C1/C3‐related novel types not previously reported. While no temporal changes in clade‐level associations were seen, there were consistent and extremely large amounts (145 unique sequences out of 153 total obtained sequences) of genotypic microvariation observed in our obtained sequences. Despite Z. sansibaricus acquiring Symbiodinium horizontally and the presence of various other Symbiodinium clades (A, G) and subclades (e.g. C15 and derived subclades) in the immediate environment, Z. sansibaricus at Sakurajima specifically associates with subclade C1/C3‐related Symbiodinium. While subclades C1/C3 have been found in a variety of different environments and are believed to be ancestral, ‘generalist’ types of Symbiodinium, C1/C3‐related clades such as seen here may be more adapted to specialized niches. We theorize that specific and year‐round association with many different types of subclade C1/C3‐related Symbiodinium helps Z. sansibaricus to survive in the fluctuating Sakurajima environment.     

Kinematics of normal locomotion—A statistical study based on T.V. data

A T.V.-computer system for the measurement and analysis of the kinematics of normal and abnormal locomotion has been developed which has the following characteristics: minimum encumberment to the subject or patient, determination of absolute coordinates of anatomical landmarks, analysis of 3 or 4 strides, and automation of data conversion and analysis. Analysis techniques have been developed which take advantage of the accuracy of the marker coordinates, and with suitable application of noise reduction filtering, permit direct calculation of velocities and accelerations (both linear and angular). Examples of some of these derived parameters vs time are presented. The results of a statistical study on a group of normal subjects walking at slow, comfortable and fast cadences yield the ranges of many velocities and accelerations not previously available.     

Properties of photoreductions by photosystem II in isolated chloroplasts. III. The effect of uncouplers on phenylenediamine shuttles across the membrane in the presence of dibromothymoquinone

In the presence of the plastoquinone antagonist dibromothymoquinone the photoreduction of ferricyanide by isolated chloroplast membranes is attributed to Photosystem II. The reaction is stimulated by the addition of phenylenediamine or C-substituted phenylenediamines (which may form a diimine on oxidation) but not of N-substituted phenylenediamines (which form a stable radical on oxidation). Phenylenediamines also restore NADP reduction (and O₂ evolution) in 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)-treated chloroplasts. In this bypassing of the inhibition site, N-substituted phenylenediamines are very effective, whereas p-phenylenediamine and C-substituted phenylenediamines are inefficient. Uncouplers exhibit a surprising effect on these systems. Even under coupling conditions uncouplers inhibit electron flow to ferricyanide mediated by phenylenediamine in the pH range 7.3–8.0, whereas the restoration of the NADP system is stimulated.

For the interpretation of the results the side of the membrane involved is considered. It is proposed that in ferricyanide reduction by Photosystem II, a phenylenediimine/diamine shuttle operates which moves reducing equivalents from the inside to the outside across the membrane. This shuttle requires a pH gradient across the membrane because of different optimal ratios of diimine/diamine inside and outside. This pH difference is abolished by the uncoupler, accounting for the observed inhibition.

The restoration of electron flow from water to NADP in DBMIB-treated chloroplasts is assumed to be a bypass of the inhibition site inside the membrane via a phenylenediamine. Because the imine/amine ratio brought about by the pH gradient is not favorable for the inside oxidation an uncoupler stimulates NADP reduction even under coupling conditions.

Also in photoreductions by Photosystem I, for example NADP reduction at the expense of P-phenylenediamine/ascorbate, a shuttle of reducing equivalents across the membrane occurs but this time from outside to inside.     

Population genomics of the filarial nematode parasite Wuchereria bancrofti from mosquitoes

Wuchereria bancrofti is a parasitic nematode and the primary cause of lymphatic filariasis – a disease specific to humans. W. bancrofti currently infects over 90 million people throughout the tropics and has been acknowledged by the world health organization as a vulnerable parasite. Current research has focused primarily on the clinical manifestations of disease and little is known about the evolutionary history of W. bancrofti. To improve upon knowledge of the evolutionary history of W. bancrofti, we whole genome sequenced 13 W. bancrofti larvae. We circumvent many of the difficulties of multiple infections by sampling larvae directly from mosquitoes that were experimentally inoculated with infected blood. To begin, we used whole genome data to reconstruct the historical population size. Our results support a history of fluctuating population sizes that can be correlated with human migration and fluctuating mosquito abundances. Next, we reconstructed the putative pedigree of W. bancrofti worms within an infection using the kinship coefficient. We deduced that there are full‐sib and half‐sib relationships residing within the same larval cohort. Through combined analysis of the mitochondrial and nuclear genomes we concluded that this is likely a results of polyandrous mating, the first time reported for W. bancrofti. Lastly, we scanned the genomes for signatures of natural selection. Annotation of putative selected regions identified proteins that may have aided in a parasitic life style or may have evolved to protect against current drug treatments. We discuss our results in the greater context of understanding the biology of an animal with a unique life history and ecology.     

Environmental Enrichments for a Group of Captive Macaws: Low Interaction Does Not Mean Low Behavioral Changes

Environmental enrichment has been widely used to improve conditions for nonhuman animals in captivity. However, there is no consensus about the best way to evaluate the success of enrichments. This study evaluated whether the proportion of time spent interacting with enrichments indicated the proportion of overall behavioral changes. Six environmental enrichments were introduced in succession to 16 captive macaws, and interaction of the animals with them as well as the behaviors of the group were recorded before and during the enrichments. All of the enrichments affected the proportions of time spent in different behaviors. Macaws interacted more with certain items (hibiscus and food tree) than with others (a toy or swings and stairs), but introduction of the enrichments that invoked the least interaction caused as many behavioral changes as those that invoked the most. Moreover, feeding behavior was only affected by the enrichment that invoked the least interaction, a change not detected by a general analysis of enrichment effects. In conclusion, little interaction with enrichment does not mean little change in behavior, and the effects of enrichments are more complex than previously considered.