Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ss-tubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils.     

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.     

Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca²⁺. The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H₂O₂) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.     

Meiotic chromosome pairing in Stethophyma grossum spermatocytes studied by a surface-spreading and silver-staining technique

Spermatocytes of Stethophyma grossum were prepared for light microscope examination by a surface-spreading and silver-staining technique adapted with very little modification from a mammalian procedure. Very clear preparations of a variety of prophase I stages were obtained which revealed many details of chromosome and nuclear organisation. These observations and preliminary observations on two other grasshopper species demonstrate the ready applicability of these techniques to Orthopteran spermatocytes. A detailed study of six pachytene stage spermatocytes gave conclusive confirmation that three bivalents achieve full pairing in male meiosis of Stethophyma grossum, the other eight bivalents being partially paired at their procentric ends only.     

Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction

Biochemical and morphological studies were performed on Novikoff hepatoma ascites cell nucleolar matrix fractions prepared by deoxyribonuclease I digestion and high-molarity salt extractions essentially according to a published method [Berezney, R., & Buchholz, L. A. (1981) Exp. Cell Res. 20, 4995-5002]. The nucleolar matrix fraction was enriched in polypeptides of molecular mass of 28, 37.5, 40, 70, 72, 110 (protein C23), and 160 kDa, compared to the nuclear fraction in which polypeptides of molecular mass of 31, 33.5, 43.5, 46, 50, 56, and 59 kDa were predominant. About one-fourth of the protein, half of the RNA, and less than 4% of the DNA originally present in the nucleoli remained in the matrix fraction. Addition of single agents such as ethylenediaminetetraacetic acid, ribonuclease A, or mercaptoethanol during preparation had no significant effect on the polypeptide composition of the nucleolar matrix fraction. However, the combination of mercaptoethanol and ribonuclease A caused most of the RNA and protein to be removed, including protein C23 and the 160-kDa polypeptide, with polypeptides in the range of Mr 30 000-50 000 remaining. Electron microscopy of nucleolar matrix fractions revealed the presence of particles similar in size to the granular elements of nucleoli. However, when ribonuclease A and mercaptoethanol were included in the procedure, only amorphous material remained. Many proteins of nucleolar preribosomal RNP particles were also associated with the nucleolar matrix fraction. RNA from the nucleolar matrix fraction was enriched in sequences from 18S and 28S ribosomal RNA. These results indicate that preribosomal RNP particles are major constituents of a nucleolar matrix fraction prepared by the deoxyribonuclease I-high-molarity salt method.     

Introduction of Exogenous Epitopes in the Variable Regions of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein: Effect on Viral Infectivity and the Neutralization Phenotype

In this study we examined whether human immunodeficiency virus type 1 (HIV-1) is equally susceptible to neutralization by a given antibody when the epitope of this antibody is introduced at different positions within the viral envelope glycoprotein (Env). To this end, we introduced two exogenous “epitope tags” at different locations within three major Env regions in two distinct HIV-1 isolates. We examined how the introduction of the exogenous epitopes affects Env expression, Env incorporation into virions, Env fusogenic potential, and viral susceptibility to neutralization. Our data indicate that even within the same Env region, the exact positioning of the epitope impacts the susceptibility of the virus to neutralization by the antibody that binds to that epitope. Our data also indicate that even if the same epitope is introduced in the exact same position on two different Envs, its exposure and, as a result, the neutralization susceptibility of the virus, can be very different. In contrast to the findings of previous studies conducted with HIV-1 isolates other than those used here, but in agreement with results obtained with simian immunodeficiency virus, we observed that tagging of the fourth variable region of Env (V4) did not result in neutralization by the anti-tag antibodies. Our data indicate that epitopes in V4 are not properly exposed within the functional HIV-1 trimeric Env spike, suggesting that V4 may not be a good target for vaccine-elicited neutralizing antibodies.The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is expressed as a heavily glycosylated peptide of approximately 160 kDa (gp160), which is cleaved intracellularly into two noncovalently associated subunits: an extracellular subunit (gp120), responsible for CD4 and coreceptor (primarily CCR5 and/or CXCR4) binding, and a transmembrane subunit (gp41) that mediates fusion between viral and host cell membranes. Based on amino acid sequence homology analysis of gp120s derived from diverse HIV-1 isolates, gp120 is divided into five “constant” regions (C1 to C5) and five “variable” regions (also called “loops,” because most of them have cysteines in the N and C termini that form disulfide bonds). Despite their extensive amino acid variability, the variable loops of gp120 play central roles during the entry of the virus into the cell, for instance, by directly or indirectly modulating the interaction of Env with coreceptor molecules on the target surfaces during virus-cell fusion. They also offer protection from neutralizing antibodies (NAbs) by various mechanisms. The variable loops themselves are targets of NAbs, and during infection, the replicating virus accumulates mutations in the variable regions that allow it to escape the action of anti-variable loop-directed NAbs, while at the same time the variable loops are positioned within the Env trimer so that they prevent, or minimize, the binding of NAbs to more-conserved epitopes, such as the receptor and coreceptor binding sites (4, 5, 12, 15, 20, 23, 25, 27, 31).HIV-1 strains display distinct neutralization phenotypes. Some isolates, such as SF162, are generally susceptible to NAbs that bind to many distinct regions of Env, including the variable regions, while other isolates, such as YU2 or JRFL, are generally resistant to neutralization by the same NAbs (1). It has been proposed that irrespective of the overall neutralizing phenotype of HIV-1 isolates, the binding of only a single antibody per Env trimer on the virion surface can lead to neutralization, when all Env trimers present on the virion surface are bound by at least one antibody (32). This important observation also implies that the epitope specificity of an antibody may not be as important for neutralization as its ability to bind to its target within the trimeric Env structure. In fact, antibodies to diverse regions of Env, such as V1, V2, V3, and the receptor and coreceptor binding sites, can all neutralize HIV-1 (1, 3, 6, 8, 10, 18, 20, 23, 25, 27, 29, 30).In many cases, a given isolate will not be equally susceptible to neutralization by NAbs that bind to different Env regions, for example, the V3 loop and the CD4-binding site (CD4-BS). Whether differences in the neutralizing potentials of two antibodies that bind to distinct epitopes on HIV-1 Env are due to differences in the binding affinities of the two antibodies or whether they occur because the viruses are intrinsically more susceptible to NAbs that bind certain epitopes and not others (i.e., the relative importance of the various regions of Env in Env function and virus neutralization sensitivity differs) is not yet fully understood. One way to address these issues is to introduce small non-HIV Env amino acid sequences (tags) that are targets of known monoclonal antibodies (MAbs) at various positions within the viral Env and to examine how the placement of the same epitope at different positions within Env affects the neutralization phenotype of the virus.Foreign epitopes have been introduced into the variable regions of HIV and simian immunodeficiency virus (SIV) Envs, and their effects on viral neutralization potential have been examined (14, 19, 22, 33). Yang and colleagues (33) introduced the FLAG epitope into the V4 regions of three HIV-1 isolates (YU2, JRFL, and HxB2) displaying distinct neutralization phenotypes in response to anti-HIV NAbs; they found that all three pseudotyped viruses were equivalently neutralized by an anti-FLAG MAb. One important implication of that study is that neutralization-resistant isolates, such as YU2 or JRFL, are not intrinsically more resistant to neutralization than more-susceptible isolates, such as HxB2, so long as the antibody binds to its epitope on the functional virion-associated Env spike. A second implication is that since the FLAG epitope was exposed in the V4 loops of all three isolates, the V4 loop could theoretically be a good target for vaccine-elicited antibodies. In contrast, Pantophlet et al. (19) introduced the HA tag into various regions of the JRCSF (neutralization-resistant) and HxB2 (neutralization-sensitive) isolates and reported that JRCSF was intrinsically more resistant than HxB2 to anti-HA antibodies. This observation implies, therefore, that some HIV-1 strains (primary, neutralization-resistant strains) have developed mechanisms that limit the accessibility of multiple Env regions, including variable regions, to antibodies developed during infection. Laird and Desrosiers (14) introduced the FLAG epitope into two positions within each of the V1, V2, and V4 loops of SIV239 and SIV316. They reported that the functionality of Env was differentially affected by the precise location of the exogenous tag sequence within the variable loops examined. Importantly, and in contrast to what was reported for the HIV-1 isolates mentioned above, the SIV239 variants containing a V4 FLAG epitope were not neutralized by an anti-FLAG MAb. It appeared, however, that the FLAG epitope was not well exposed on the trimeric Env when introduced into the V4 loop of SIV but was exposed when introduced into the V1 loop of the same virus. Potentially, this means that the V4 loop is differentially exposed in the context of the HIV-1 and SIV Envs.The FLAG epitope (DYKDDDDK) is highly charged. Therefore, it is possible that the effect on Env function and epitope exposure could differ if a different exogenous epitope were inserted instead of FLAG. Here we examined the effect of variable loop tagging on the Env functions and viral neutralization phenotypes of two primary HIV-1 clade B isolates, SF162 (CCR5 tropic) and SF33 (CXCR4 tropic), using two exogenous epitopes (FLAG and hemagglutinin [HA] tags) positioned at multiple locations within the V1, V2, and V4 loops. By placing the same tag in several regions within each loop, we investigated the accessibilities of various parts of the same loop to a given NAb. By using two tags that differ significantly in amino acid composition (FLAG tag, DYKDDDDK; HA tag, YPYDVPDYA), we aimed at distinguishing between the effects of amino acid composition and the positioning of the tag on Env function and overall epitope exposure. Finally, identical evaluations of R5 and X4 Envs may provide information about the relative roles played in neutralization by variable loops in Envs displaying distinct coreceptor usage. We report that both the amino acid sequence and the position of the tag within and among the variable loops greatly affected the functionality of Env. In contrast to previous observations made with other HIV-1 Envs (33) but in agreement with what was reported for the SIV239 Env (14), we observed that tagging of the V4 loops of SF162 and SF33 did not render these isolates susceptible to neutralization by the corresponding anti-tag MAbs.     

Middle phalanx skeletal morphology in the hand: Can it predict flexor tendon size and attachments?

Specific sites on the palmar diaphysis of the manual middle phalanges provide attachment for the flexor digitorum superficialis (FDS) tendon. It has been assumed in the literature that lateral palmar fossae on these bones reflect locations for these attachments and offer evidence for relative size of the flexor tendon. This assumption has led to predictions about relative FDS muscle force potential from sizes of fossae on fossil hominin middle phalanges. Inferences about locomotor capabilities of fossil hominins in turn have been drawn from the predicted force potential of the flexor muscle. The study reported here provides a critical first step in evaluating hypotheses about behavioral implications of middle phalangeal morphology in fossil hominins, by testing the hypothesis that the lateral fossae reflect the size of the FDS tendon and the location of the terminal FDS tendon attachments on the middle phalanx. The middle phalangeal region was dissected in 43 individuals from 16 primate genera, including humans. Qualitative observations were made of tendon attachment locations relative to the lateral fossae. Length measurements of the fossae were tested as predictors of FDS tendon cross-sectional area and of FDS attachment tendon lengths. Our results lead to the conclusion that the hypothesis must be rejected, and that future attention should focus on functional implications of the palmar median bar associated with the lateral fossae.     

Conserved Glu40 and Glu433 of the biotin carboxylase domain of yeast pyruvate carboxylase I isoenzyme are essential for the association of tetramers

The native form of pyruvate carboxylase is an alpha4 tetramer but the tetramerisation domain of each subunit is currently unknown. To identify this domain we co-expressed yeast pyruvate carboxylase 1 isozyme (Pyc1) with an N-terminal myc tag, together with constructs encoding either the biotin carboxylase (BC) domain or the transcarboxylase-biotin carboxyl carrier domain (TC-BCC), each with an N-terminal 9-histidine tag. From tag-affinity chromatography experiments, the subunit contacts within the tetramer were identified to be primarily located in the 55 kDa BC domain. From modelling studies based on known structures of biotin carboxylase domains and subunits we have predicted that Arg36 and Glu433 and Glu40 and Lys426, respectively, are involved pairwise in subunit interactions and are located on opposing subunits in the putative subunit interface of Pyc1. Co-expression of mutant forms with wild type Pyc1 showed that the R36E mutation had no effect on the interaction of these subunits with those of wild type Pyc1, while the E40R, E433R and R36E:E433R mutations caused severe loss of interaction with wild type Pyc1. Ultracentrifugal analysis of these mutants when expressed and purified separately indicated that the predominant form of E40R, E433R and R36R:E433R mutants is the monomer, and that their specific activities are less than 2% of the wild type. Studies on the association state and specific activity of the R36E mutant at different concentrations showed it to be much more susceptible to tetramer dissociation and inactivation than the wild type. Our results suggest that Glu40 and Glu433 play essential roles in subunit interactions.     

Functional gamma delta T-lymphocyte defect associated with human immunodeficiency virus infections.

BACKGROUND: Antiviral cellular immune responses may influence immunological homeostasis in HIV-infected persons. Recent data indicate that V gamma 9/V delta 2 T lymphocytes display potent cytotoxic activities against human cells infected with certain viruses including HIV. Understanding the role of gamma delta T cells in the course of HIV infection may be helpful for designing novel treatment strategies for HIV-associated disorders. MATERIALS AND METHODS: The constitutive recognition of Daudi cells and monoethyl pyrophosphate (Etpp) by peripheral blood V gamma 9/V delta 2 T cells was assessed using a proliferation assay. The cytotoxicity of Daudi-stimulated lymphocyte populations was measured by chromium release assays. The HIV infectivity for gamma delta T cell clones was determined by measuring the levels of HIV p24 in cell supernatants. The effect of in vitro HIV-infection on cytokine mRNA production by gamma delta T cell clones was assessed by PCR. RESULTS: The constitutive proliferative responses of peripheral blood V gamma 9/V delta 2 T cells and the lytic functions of Daudi-expanded lymphoid cells from HIV+ persons were substantially diminished in comparison with those of HIV-seronegative persons. These alterations were present in asymptomatic HIV+ persons prior to substantial alpha beta CD4+ T cell loss. Productive HIV infection of gamma delta T cells in vitro had no measurable effect either on their proliferative response to Daudi stimuli or on the expression of cytokine mRNAs for IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-13. CONCLUSIONS: The constitutive responsiveness of V gamma 9/V delta 2 T lymphocytes to Daudi and Etpp is severely altered in HIV+ persons. HIV infection of gamma delta T cells in vitro does not substantially change their cytokine expression or antigenic response.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.