<Emphasis Type="Italic">“Pseudoalteromonas januaria”</Emphasis> SUT 11 as the Source of Rare Lipodepsipeptides

The marine bacterium “Pseudoalteromonas januaria” SUT 11 isolated from a seawater sample produced the rare cell-bound cyclic lipodepsipeptides A/A′, B/B′, and C/C′. The matrix-assisted laser desorption/ionization mass spectra indicated that one bromine atom presented in the peptides B/B′ and C/C′, whereas the component A/A′ contained no bromine atom. The acyldepsipeptides A/A′–C/C′ have an identical amino acid sequence, Thr-Val-Asn-Asn-Leu/allo-Ile, but differed in C-terminal amino acid and acyl moieties. Peptides A–C have Leu as a C-terminal amino acid, whereas peptides A′-C′ have allo-Ile. Acyl moieties in peptides A/A′, B/B′, and C/C′ have been found to consist of 11-(4′-hydroxyphenyl)-undeca-2,4,6,8,10-pentaenic acid, 9-(3′-bromo-4′-hydroxyphenyl)-nona-2,4,6,8-tetraenic acid, and 11-(3′-bromo-4′-hydroxyphenyl)-undeca-2,4,6,8,10-pentaenic acid, respectively. The structure of a main pair of peptides B/B′ with molecular masses 843/845 Da has been determined by means of ultraviolet, infrared, and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that tandem nano-electrospray ionization mass spectrometry is a very efficient way for the fast and sensitive investigation of lipopeptides A/A′ and C/C′ with molecular masses 791 and 869/871 Da, respectively, which have been isolated in small amounts.     

Developmental interdeterminacy in embryos of the leech Helobdella triserialis

In embryonic development of the leech Helobdella triserialis, each of the four paired positionally identifiable, ectodermal teloblasts (N, O, P, and Q) generates a bandlet of blast cell progeny that merges with ipsilateral bandlets into a germinal band. Left and right germinal bands coalesce into the germinal plate which gives rise to the segmental tissues of the leech and wherein the progeny of each teloblast generate a characteristic pattern of epidermal and neuronal cells. Experiments reported here show that the positionally identified O teloblast sometimes generates the P pattern and vice versa. The reversal of these teloblasts' generative identities was shown to correspond to the formation of chiasmata by their blast cell bandlets, so that the positions of their bandlets in the germinal band are reversed as well. Thus it is the position of the bandlet in the germinal band, rather than the position of the parent teloblast, which correlates with the fate of o and p blast cells. Moreover, two types of ablation experiments have shown that, in the absence of generative P teloblast progeny, those cells which would normally generate the O pattern take on a new fate and give rise to the P pattern in the nervous system, both at the gross pattern level in the segmental ganglia, and at the level of identified neurons in the peripheral nervous system. If related, these phenomena suggest that the O and P teloblasts, which derive from the symmetric cleavage of the OP proteloblasts, have a common developmental pluripotency. And in that case, the fates of their progeny are determined hierarchically on the basis of relative position in the nascent germinal band, with P-type fate being preferred.     

Diversity in subcellular targeting of the PP2A B′η subfamily members

Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase comprising a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The B subunits are believed to be responsible for substrate specificity and localization of the PP2A complex. In plants, three families of B subunits exist, i.e. B (B55), B′, and B′′. Here, we report differential subcellular targeting within the Arabidopsis B′η subfamily, which consists of the close homologs B′η, B′θ, B′γ and B′ζ. Phenotypes of corresponding knockouts were observed, and particularly revealed delayed flowering for the B′η knockout. The B′ subunits were linked to fluorescent tags and transiently expressed in various tissues of onion, tobacco and Arabidopsis. B′η and B′γ targeted the cytosol and nucleus. B′ζ localized to the cytoplasm and partly co-localized with mitochondrial markers when the N-terminus was free. Provided its C-terminus was free, the B′θ subunit targeted peroxisomes. The importance of the C-terminal end for peroxisomal targeting was further confirmed by truncation of the C-terminus. The results revealed that the closely related B′ subunits are targeting different organelles in plants, and exemplify the usage of the peptide serine–serine–leucine as a PTS1 peroxisomal signaling peptide.     

Micromere fate maps in leech embryos: lineage-specific differences in rates of cell proliferation

Stereotyped early cleavages in glossiphoniid leech embryos yield 25 micromeres, along with 3 macromeres and 10 teloblasts. The micromeres generate prostomial tissues and also give rise to most of the squamous epithelium of a provisional integument that spreads epibolically from the animal pole, covering the rest of the embryo during germinal plate formation.We systematically injected individual micromeres with fluorescent cell lineage tracers at the time of their birth and quantitatively mapped the contributions of all these cells to the late stage 7 embryo, a time in development that is early in the epibolic expansion. At this time, micromere derivatives comprise two types of cells: squamous epithelial (superficial) cells that cover the germinal bands and the region of the animal cap between the germinal bands; and underlying (deep) cells that are confined to the distal ends of the germinal bands and in the area between their distal ends. We find that individual micromeres contribute clones of deep and/or superficial progeny that are stereotyped with respect to both numbers and types of cells in the clone and the domains that they occupy. The N teloblasts also contribute cells to the squamous epithelium.We find significant differences in the rate of cell proliferation between different micromere clones. These differences appear to reflect lineage-specific traits, since there is little or no regulation of cell number after ablation of individual micromeres.     

Evolutionary implications of the mode of D quadrant specification in coelomates with spiral cleavage

In annelids, molluscs, echiurans and sipunculids the establishment of the dorsal-ventral axis of the embryo is associated with D quadrant specification during embryogenesis. This specification occurs in two ways in these phyla. One mechanism specifies the D quadrant via the shunting of a set of cytoplasmic determinants located at the vegetal pole of the egg to one blastomere of the four cell stage embryo. In this case, at the first two cleavages of embryogenesis there is an unequal distribution of cytoplasm, generating one macromere which is larger than the others at the four cell stage. The D quadrant can also be specified by a contact mediated inductive interaction between one of the macromeres at the vegetal pole with micromeres at the animal pole of the embryo. This mechanism operates at a later stage of development than the cytoplasmic localization mechanism and is associated with a pattern of cleavage in which the first two cleavages are equal. An analysis of the phylogenetic relationships within these phyla indicates that the taxa which determine the D quadrant at an early cleavage stage by cytoplasmic localization tend to be derived and lack a larval stage or have larvae with adult characters. Those taxa where the D quadrant is specified by induction include the ancestral groups although some derived groups also use this mechanism. The pulmonate mollusc Lymnaea uses an inductive mechanism for specifying the D quadrant. In these embryos each of the four vegetal macromeres has the potential of becoming the D macromere; however under normal circumstances one of the two vegetal crossfurrow macromeres almost invariably becomes the D quadrant. Experiments are described here in which the size of one of the blastomeres of the four cell stage Lymnaea embryo is increased; this macromere invariably becomes the D quadrant. These experiments suggest that developmental change in relative blastomere size during the first two cleavages in spiralian embryos that normally cleave equally may have provided a route that has led to the establishment of the cytoplasmic localization mechanism of D quadrant formation.     

Relational reasoning with derived comparative relations: A novel model of transitive inference

A behavior-analytic model of transitive inference (TI) as relational reasoning with derived comparative relations is outlined. Following nonarbitrary relational training and testing to establish contextual functions of “more than” (>) and “less than” (<) for two abstract stimuli, two groups of participants learned a series of contextually controlled more than or less than relations (All-More: E > D > C > B > A; All-Less: A < B < C < D < E). On meeting the training criterion, inferential tests were presented to both groups involving mutually entailed relations (All-More: A < B, B < C, C < D and D < E; All-Less: B > A, C > B, D > C and E > D) and one-step (A < C, B < D, C < E, C > A, D > B and E > C) and two-step (A < D, B < E, D > A and E > B) combinatorially entailed relations. Performance accuracy on the trained and inferential tasks was uniformly high across both groups, with no significant differences observed. In both groups, however, performance accuracy differed significantly on one-step and two-step combinatorially entailed tasks involving the same or different relation to that trained. The present findings demonstrate complex relational reasoning with derived comparative relations, replicate several key effects from the literature on TI and have potential implications for the development of a contemporary behavior-analytic account of TI.     

The production and elimination of supernumerary blast cells in the leech embryo

 Different species of leech vary greatly in body size but all have 32 body segments. It is unclear how the development of this precise number of segments is regulated, although it is known that the teloblasts of the early leech embryo initially produce more than the required numbers of segment founder cells (blast cells). We used fluorescent dextrans to show that the M teloblast of the Helobdella robusta embryo produces a variable number of additional (supernumerary) cells. These cells fail to enter the germinal band (which contains cells of all lineages and gives rise to the adult leech), but detach from its posterior end and disappear. Our observations suggest that some suffer an increase in membrane permeability while others fuse with the M teloblasts, but that they do not undergo apoptosis. The supernumerary cells of different lineages detach from the germinal band at different times, suggesting that detachment is not triggered by a global signal acting simultaneously on all lineages. We tested the hypothesis that the elimination of the supernumerary m blast cells results from a requirement of m blast cells for close interactions with cells of the other lineages for their survival, a condition that would not be achieved by the last-born m blast cells that fail to enter the germinal band. We cultured isolated M teloblasts and found that they do produce blast cells that themselves divide, indicating that cells of the M lineage can survive in the absence of any interactions with cells of the other lineages. Received: 17 August 1998 / Accepted: 20 November 1998     

The ‘organizing’ role of the D quadrant in an equal-cleaving spiralian,Lymnaea stagnalis as studied by UV laser deletion of macromeres at intervals between third and fourth quartet formation

Summary

In the spiralian embryos studied which display unequal-cleavage at the first two cleavages (either by a polar lobe or an asymmetric cleavage mechanism) the D quadrant is determined at the four cell stage by an unequal segregation of cytoplasmic stuffs. The normal formation of eyes, foot, and shell by overlying micromeres in these forms requires the inductive interaction with the D quadrant before the formation of the third quartet of micromeres. In equal-cleaving spiralians the D quadrant (3D macromere) becomes determined as a result of inductive interactions with first quartet derivatives (animal-vegetal interaction) sometime after the production of the third quartet of micromeres. This paper investigates the exact timing of D quadrant determination and the inductive role of third-order macromeres on the development of micromere derived structures in an equal-cleaving spiralian. Deletions of third-order macromeres, and their derivatives, were performed without rupturing the egg capsule membrane of the Lymnaea embryo with a UV laser microbeam. Virtually normal snails were produced when the 3A, 3B, 3C, or 4D macromere was irradiated. Juvenile snails lacking all mesodermal structures but possessing eyes, foot, and shell were obtained when the mesentoblast (4d) or its progenitor (3D) were deleted. Furthermore, ‘mesoderm-less’ snails were produced by deleting one of the two possible 3D candidates (cross furrow macromeres) as early as 20 min after third quartet formation. These results indicate that the 3D macromere begins to become determined at, or soon after, animal-vegetal interaction; before the 3D macromere becomes visibly distinguishable from the 3B macromere. The results also demonstrate that normal pattern formation in the overlying micromeres does not require the ‘prolonged’ interaction with an asymmetrically positioned 3D macromere. Possible adhesive differences between the 3D macromere and the remaining three macromeres are also revealed.     

Lesions in thighs from hunted Brown Hares (<Emphasis Type="Italic">Lepus europaeus</Emphasis>) and microflora under vacuum-packaging storage

In two surveys, thighs of a total of 137 hunted hares were tested for the presence of intramuscular shots and femur fractures, which were detected in 42.7% and 29.2% of 274 thighs, respectively. Femur fractures were significantly associated with the presence of intramuscular shots. In the second survey (46 hares), 92 thighs were grouped into three categories, “A” (no fractures, no intramuscular shot), “B” (one intramuscular shot), and “C” (multiple shots and hematoma), with 49.0%, 33.6%, and 17.4%, respectively. Category “C” was found unfit for human consumption. During 7-day storage of vacuum-packed “A” and “B” thighs, total aerobic counts increased from initially 3.3 ± 0.3 (mean ± SD) and 4.1 ± 0.6 log cfu/g by ca. 2 log units when stored at 3–4°C, whereas the increase was clearly <1 log unit at 0°C. In comparison to temperature, differences between “A” and “B” category were less pronounced. Similar dynamics were observed for Enterobacteriaceae. In all categories, muscle pH values (mean = 5.83) were similar. It is concluded that storage at temperatures of ca. 4°C, although in compliance with EU legislation, does not afford keeping microbial contaminants in check, and thus will not preserve microbiological quality of vacuum-packed hare meat.     

An investigation of the specification of unequal cleavages in leech embryos.

Leech embryos develop via stereotyped cell divisions, many of which are unequal. The first division generates identifiable cells, blastomeres AB and CD, which normally follow distinct developmental pathways. When these two cells are dissociated and cultured in isolation, their fates remain distinct and are reminiscent of normal development, but their typical cleavage patterns are disrupted; cell AB undergoes relatively few cell divisions, giving rise to a variable number of macromeres and micromeres, while cell CD cleaves many times, usually forming a poorly organized set of macromeres, embryonic stem cells (teloblasts), and micromeres. We have investigated the hypothesis that the abnormal cleavage pattern of isolated CD blastomeres is due to removal of mechanical constraints normally imposed by cell AB. We find that when cell CD is constrained in vitro to mimic its in vivo shape, it cleaves more normally.     

What studies of turbellarian embryos can tell us about the evolution of development mechanisms

In spiralian embryos determination of the axes of bilateral symmetry is associated with D quadrant specification. This can occur late through equal cleavage and cell interactions (conditional specification) or by the four-cell stage through unequal cleavage and cytoplasmic localization (autonomous specification). Freeman & Lundelius (1992) suggest that in spiralian coelomates the former method is ancestral and the latter derived, with evolutionary pressure to shorten metamorphosis resulting in early D quadrant determination through unequal cleavage and appearance of adult features in the larvae. Because of the key phylogenetic position of the turbellarian platyhelminthes, understanding the method of axis specification in this group is important in evaluating the hypothesis. Polyclad development, with equal quartet spiral cleavage, is believed to represent the most primitive condition among living turbellarians and has been examined experimentally in Hoploplana inquilina. Blastomere deletions at the two and four-cell stage produce larvae that are abnormal in morphology and symmetry, indicating that early development is not regulative, and also establish that the embryo does not have an invariant cell lineage. Deletions of micromeres and macromeres at the eight-cell stage indicate that cell interactions are involved in dorso-ventral axis determination, with cross-furrow macromeres playing a more significant role than non-cross-furrow cells. The results support the idea that conditional specification is the primitive developmental mode that characterized the common ancestor of the turbellarians and spiralian coelomates. Evolutionary trends in development in polyclads and other turbellarian orders are discussed.     

Theoretical study of cyclohexane hydroxylation by three possible isomers of [FeIV(O)(R-TPEN)]2+: does the pentadentate ligand wrapping around the metal center differently lead to the different stability and reactivity?

Density functional theory calculations have been carried out to elucidate the mechanism of cyclohexane hydroxylation by three possible isomers of [Fe^IV(O)(N-R-N,N′,N′-tris(2-pyridylmethyl)ethane-1,2-diamine)]²⁺ (R is methyl or benzyl) (Klinker et al. in Angew Chem Int Ed 44:3690–3694, 2005). The calculations offer a mechanistic view and reveal the following features: (a) all the three isomers possess triplet ground states and low-lying quintet excited states, (b) the relative stability follows the order isomer A > isomer B > isomer C, in agreement with the conclusions of Klinker et al., (c) the theoretical investigations provide a rationale to explain the interconversion of the three isomers, (d) the reaction pathways of the C–H hydroxylation are initiated by a hydrogen-abstraction step, and (e) the three isomers react with cyclohexane via two-state-reactivity patterns on competing triplet and quintet spin-state surfaces. As such, in the gas phase, the relative reactivity exhibits the trend isomer B > isomer A, while at the highest level, B2//B1 with zero point energy and solvation corrections, the relative reactivity follows the order isomer B > isomer A > isomer C. Thus, the calculated reaction pathway shows that pyridine rings perpendicular to the Fe–O axis result in more reactive species, and a pyridine ring coordinated trans to the oxygen atom leads to the least reactive isomer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Cell lineage and segmentation in the leech

Segments in the leech arise by the proliferation of longitudinally arrayed bandlets of blast cells derived from ten identifiable embryonic stem cells, two M, two N, four O/P and two Q teloblasts. In each bandlet, older blast cells lie ahead of those born later. By using microinjected cell lineage tracers it was shown previously that the teloblasts give rise to characteristic cell patterns made up of segmentally iterated complements of progeny designated as M, N, O, P and Q kinship groups. When a teloblast is injected after it has begun generating blast cells, a boundary is observed later in development between anterior, unlabelled progeny of blast cells produced before injection and posterior, labelled progeny of blast cells produced after injection. We have examined such boundaries in detail to establish the precise relationship between blast cell clones and segments, with the following conclusions: (i) in the M, O and P cell lines, one blast cell generates one segmental complement of progeny, but serially homologous blast clones intermix so that no segment boundaries can be defined based on primary blast cell clones; (ii) in the N and Q cell lines, two blast cells are required to generate a complete segmental complement of progeny; (iii) in the process of forming the germinal plate, cells derived from the N and Q teloblasts move past those derived from the M and O/P teloblasts, so that consegmental blast cell clones do not come into register until well after the establishment of segmentally iterated units within each bandlet.     

Conserved mechanism of dorsoventral axis determination in equal-cleaving spiralians

Many members of the spiralian phyla (i.e., annelids, echiurans, vestimentiferans, molluscs, sipunculids, nemerteans, polyclad turbellarians, gnathostomulids, mesozoans) exhibit early, equal cleavage divisions. In the case of the equal-cleaving molluscs, animal-vegetal inductive interactions between the derivatives of the first quartet micromeres and the vegetal macromeres specify which macromere becomes the 3D cell during the interval between fifth and sixth cleavage. The 3D macromere serves as a dorsal organizer and gives rise to the 4d mesentoblast. Even though it has been argued that this situation represents the ancestral condition among the Spiralia, these inductive events have only been documented in equal-cleaving molluscs. Embryos of the nemertean Cerebratulus lacteus also undergo equal, spiral cleavage, and the fate map of these embryos is similar to that of other spiralians. The role of animal first quartet micromeres in the establishment of the dorsal (D) cell quadrant was examined in C. lacteus by removing specific combinations of micromeres at the eight-cell stage. To follow the development of various cell quadrants, one quadrant was labeled with DiI at the four-cell stage, and specific first quartet micromeres were removed from discrete positions relative to the location of the labeled quadrant. The results indicate that the first quartet is required for normal development, as removal of all four micromeres prevented dorsoventral axis formation. In most cases, when either one or two adjacent first quartet micromeres were removed from one side of the embryo, the cell quadrant on the opposite side, with its macromere centered under the greatest number of the remaining animal micromeres, ultimately became the D quadrant. Twins containing duplicated dorsoventral axes were generated by removal of two opposing first quartet micromeres. Thus, any cell quadrant can become the D quadrant, and the dorsoventral axis is established after the eight-cell stage. While it is not yet clear exactly when key inductive interactions take place that establish the D quadrant in C. lacteus, contacts between the progeny of animal micromeres and vegetal macromeres are established during the interval between the fifth and sixth round of cleavage divisions (i.e., 32- to 64-cell stages). These findings argue that this mechanism of cell and axis determination has been conserved among equal-cleaving spiralians.     

Comparative Studies of Seed Germination of two Newly Isolated Lines of Grand Rapids Lettuce

Two lines of Grand Rapids lettuce were isolated by self-pollinationof single plants. Seeds of one line, designated ‘A’,germinate in the dark at 25 °C. Seeds of the second line,designated ‘B’ germinate in a manner typical oftheir variety. The lines differ in sensitivity to red (R) andfar-red (FR) light, in the ability of their seeds to overcomeresistance of an external osmotically active medium, in theresponse of mature seeds to abscisic acid, and in the responseof young seeds to gibberellin and benzyl adenine. ‘A’seeds germinate faster than ‘B’ seeds in the darkor after R or FR irradiation.     

The Extraction, Solubility, and Characterization of Two Groups of Barley Storage Polypeptides

This paper reports further studies on the characteristics ofthe storage protein fraction (hordein) of barley. Hordein consistsof two groups of polypeptides (termed ‘B’ and ‘C’)coded by two separate but linked loci. Whereas the ‘C’polypeptides are readily soluble and extracted in 60% (v/v)ethanol at room temperature, the ‘B’ group is moresoluble in, and therefore more efficiently extracted by, 50%(v/v) propan-1-ol or 45% (v/v) propan-2-ol at elevated temperaturesand in the presence of 2-mercaptoethanol. However, the mostefficient conditions for hordein extraction (50% propan-1-ol+ 2% (v/v) 2-mercaptoethanol at 60 °C) also extract somecontaminating non-hordein polypeptides resulting in an apparentlyincreased lysine content of the hordein fraction. Amino acid analysis of the purified ‘B’ and ‘C’hordein groups shows that, whereas ‘C’ hordein containsmore glutamate + glutamine, proline, and phenylalanine than‘B’ hordein, it contains only traces of lysine andsulphur amino acids in contrast to ‘B’ hordein whichcontains 0·5% lysine 0·6% methionine, and 2·5%cysteine. Equilibrium sedimentation analyses carried out on the purified‘B’ and ‘C’ groups indicates that thepreparations were reasonably monodisperse with molecular weightsof approximately 32 000 and 52 000 respectively. These valuesare considerably lower than those previously determined by SDS-PAGE.     

Improved accuracy in measuring one-bond and two-bond <Superscript>15</Superscript>N,<Superscript>13</Superscript>C<Superscript>α</Superscript> coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

An extension to HN(CO-α/β-N,C^α-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants ¹ J _{N′(i)Cα(i)}, ² J _HN(i)Cα(i), ² J _{Cα(i−1)N′(i)}, and ³ J _{Cα(i−1)HN(i)} in ¹⁵N,¹³C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the ² J _CαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both ² J _CαN′ and ¹ J _N′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.     

Cell-cell interactions determine the dorsoventral axis in embryos of an equally cleaving opisthobranch mollusc

Dorsoventral polarity in molluscan embryos can arise by two distinct mechanisms, where the mechanism employed is strongly correlated with the cleavage pattern of the early embryo. In species with unequal cleavage, the dorsal lineage, or "D quadrant", is determined in a cell-autonomous manner by the inheritance of cytoplasmic determinants. However, in gastropod molluscs with equal cleavage, cell-cell interactions are required to specify the fate of the dorsal blastomere. During the fifth cleavage interval in equally cleaving embryos, one of the vegetal macromeres makes exclusive contacts with the animal micromeres, and this macromere will give rise to the mesodermal precursor cell at the next division, thereby identifying the dorsal quadrant. This study examines D-quadrant determination in an equally cleaving species from a group of previously uninvestigated gastropods, the subclass Opisthobranchia. Blastomere ablation experiments were performed on embryos of Haminoea callidegenita to (i) determine the developmental potential of macromeres before and after fifth cleavage, and (ii) examine the role of micromere-macromere interactions in the establishment of bilateral symmetry. The results suggest that the macromeres are developmentally equivalent prior to fifth cleavage, but become nonequivalent soon afterward. The dorsoventral axis corresponds to the displacement of the micromeres over one macromere early in the fifth cleavage interval. This unusual cellular topology is hypothesized to result from constraints imposed on micromere-macromere interactions in an embryo that develops from a large egg and forms a stereoblastula (no cleavage cavity). Ablation of the entire first quarter of micromeres results in embryos which remain radially symmetrical in the vegetal hemisphere, indicating that micromere-macromere interactions are required for the elaboration of bilateral symmetry properties. Therefore, inductive interactions between cells may represent a general strategy for dorsoventral axis determination in equally cleaving gastropods.     

The role of animal-vegetal interaction with respect to the determination of dorsoventral polarity in the equal-cleaving spiralian,Lymnaea palustris

Summary Spirally cleaving embryos in which the first two cleavages generate four equal-sized blastomeres remain radially symmetrical along their animal-vegetal axis until the interval between third and fourth quartet formation. At this time animal micromeres and vegetal macromeres contact each other as they elongate and occlude the central, fluid-filled cleavage cavity. The overlying micromeres focus their contacts onto one of the four macromeres, the presumptive 3D macromere, as it elongates to a central position within the embryo. We tested the hypothesis that this animal-vegetal interaction was causally involved in the determination of the symmetry properties in both the animal and vegetal hemispheres by reversibly inhibiting animal-vegetal contact at the 24 cell stage with cytochalasin-B. Embryos remained hollow throughout the treatment period and animal-vegetal interaction did not occur. After treatment, blastomere elongation occurred but no D quadrant macromere appeared and the vegetal hemisphere remained radialized. On the basis of cleavage and ciliation patterns of first quartet derivatives, treated embryos remained fully or partially radialized, showing a strong tendancy to develop as ventral quadrants. These results show that the quadrants of this equal-cleaving spiralian are not definitively determined until after the 24 cell stage and that animal-vegetal interaction is required for D quadrant determination. The mechanisms of symmetrization in the animal and vegetal hemispheres of equal-cleaving spiralians is also discussed.     

Heteromeric complexes of heat shock protein 70 (HSP70) family members,including Hsp70B′, in differentiated human neuronal cells

Human neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have been termed “protein misfolding disorders.” Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B′ which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B′, Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B′ also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members.