The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity

Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a central but enigmatic role in cell division. This protein associates with FtsB and FtsL, which, like FtsQ, are bitopic inner membrane proteins with a large periplasmic domain (denoted FtsQ_p, FtsB_p, and FtsL_p) that is indispensable for the function of each protein. Considering the vital nature and accessible location of the FtsQBL complex, it is an attractive target for protein-protein interaction inhibitors intended to block bacterial cell division. In this study, we expressed FtsQ_p, FtsB_p, and FtsL_p individually and in combination. Upon co-expression, FtsQ_p was co-purified with FtsB_p and FtsL_p from E. coli extracts as a stable trimeric complex. FtsB_p was also shown to interact with FtsQ_p in the absence of FtsL_p albeit with lower affinity. Interactions were mapped at the C terminus of the respective domains by site-specific cross-linking. The binding affinity and 1:1:1 stoichiometry of the FtsQ_pB_pL_p complex and the FtsQ_pB_p subcomplex were determined in complementary surface plasmon resonance, analytical ultracentrifugation, and native mass spectrometry experiments.     

Population and infection dynamics of Daubaylia potomaca (Nematoda: Rhabditida) in Helisoma anceps

Daubaylia potomaca is an unusual parasite for several reasons. Specifically, it has a direct life cycle in which it uses a planorbid snail, Helisoma anceps , as the definitive host. In addition, adult females have been shown to be both the infective stage and the only stage documented to be shed from a live, infected host. Finally, adults, juveniles, and eggs have been observed in all tissues and blood spaces of the host, suggesting the parasite consumes and actively migrates through host tissue. The present study examined the population and infection dynamics of D. potomaca in Mallard Lake, a 4.9-ha public access pond in the Piedmont region of North Carolina. In particular, the study examined the role of seasonality on the prevalence and mean intensity of infection of D. potomaca in the snail host. Data collected from August 2008 to October 2009 suggest that prevalence and mean intensity were inversely related in the spring and fall. Prevalence in fall 2008 was 10.3% but increased to 47.3% in spring 2009. Conversely, intensity was high in fall 2008 at 52.4 ± 8.9 worms/infected host but dropped to 3.1 ± 0.3 worms/infected host in spring 2009. During the same time, the parasites within the snails went from highly aggregated populations in the fall to a less aggregated distribution in the spring. It is hypothesized that D. potomaca induces mortality of the snail hosts during the winter, followed by a rapid recruitment event of the nematodes by the snail population after torpor.     

High biological activity of the synthetic replicates of somatostatin-28 and somatostatin-25

We have isolated form extracts of ovine hypothalami two molecules characterized as somatostatin-28 and somatostatin-4-28 (referred to as somatostatin-25). They were reproduced by solid hase synthesis. In equimolar ratio and depending upon the experimental conditions, synthetic somatostatin-28 ans somatostatin-25 are 3-14 times more potent than somatostatin-14 to inhibit the basal in vitro secretion of growth hormone or as stimulated by prostaglandin (PGE2). In early studies in vivo, somatostatin-28 and somatostatin-25 are also more potent than somatostatin-14 in inhibiting the secretion of growth hormone acutely stimulated in the rat by injection of morphine; somatostatin-28 is also longer-acting than somatostatin-14. These results suggest that somatostatin-14, as originally isolated, is a biologically active fragment of a larger molecule of greater specific activity; it should be considered as another form of somatostatin with high biological activity present in some tissues and likely secreted y the tissues along with somatostatin-14 and possibly other somatostatin-peptides of diverse sizes.     

Chemotaxis ofAeromonas hydrophila to the surface mucus of fish

Isolates ofAeromonas hydrophila from various sources show different chemotactic responses to mucus from the surface of freshwater fish. Some isolates were nonchemotactic to fish surface mucus. Isolates ofA. hydrophila from fish lesions had a significantly higher chemotactic index than isolates ofA. hydrophila from water. Maximum chemotactic responses occurred more often to diluted fish mucus than to undiluted samples. Fish which were experimentally stressed did not produce mucus that was more or less chemotactic than that of unstressed fish. Fish with red-sore lesions produced surface mucus which was not chemotactic toA. hydrophila. Differences between fish, for any isolate, were also not significant. The chemotactic substance(s) in fish mucus has a molecular weight of approximately 100,000 and did not appear to be labile when heated to 56°C.     

Differential effects of NgCAM and N-cadherin on the development of axons and dendrites by cultured hippocampal neurons

A fundamental step in neuronal development is the acquisition of a polarized form, with distinct axons and dendrites. Although the ability to develop a polarized form appears to be largely an intrinsic property of neurons, it can be influenced by environmental cues. For example, in cell cultures substrate and diffusible factors can enhance and orient axonal development. In this study we examine the effects of growth on each of two cell adhesion molecules (CAMs), NgCAM and N-cadherin, on the development of polarity by cultured hippocampal neurons. We find that although the same pattern of development occurs on control substrates and the CAMs, the CAMs greatly accelerate the rate and extent of development of axons—axons form sooner and grow longer on the CAMs than on the control substrate. In contrast, the CAMs have opposite effects on dendritic development—N-cadherin enhances, but NgCAM reduces dendritic growth compared to control. These results provide further evidence that the development of polarity is largely determined by a cell-autonomous program, but that environmental cues can independently regulate axonal and dendritic growth.     

Functional Characterization Improves Associations between Rare Non-Synonymous Variants in CHRNB4 and Smoking Behavior

Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration–response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC₅₀ (β = −0.67, r² = 0.017, P = 2×10⁻⁴) or by response to low nicotine (β = −0.29, r² = 0.02, P = 6×10⁻⁵) when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted (β = −0.04, r² = 0.0009, P = 0.54). These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence.     

Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins

Two forms of inhibin (A and B), gonadal polypeptide hormones that selectively suppress the secretion of FSH from the anterior pituitary, have been characterized from the porcine and human species, each being composed of a common alpha-chain and one of two distinct, but homologous beta-chains, i.e. alpha beta A and alpha beta B. Using cDNAs encoding the porcine inhibin subunits we have cloned and sequenced the cDNAs encoding the alpha, beta A, and beta B chains of rat ovarian inhibin. Northern analyses of rat testicular RNA with rat ovarian cDNA probes show the presence of mRNAs encoding alpha and beta B chains, but no detectable mRNA encoding the beta A chain under our experimental conditions. This suggests that there may be specific and distinct physiological roles for inhibins A and B. In addition, if there is no extratesticular source of beta A mRNA, then the male rat may be devoid of the stimulators of the secretion of FSH, i.e. activin (beta A beta B) and homoactivin A (beta A beta A), which are derived from the beta subunits of the two inhibins.     

Mutations in the Small GTPase Gene RAB39B Are Responsible for X-linked Mental Retardation Associated with Autism, Epilepsy, and Macrocephaly

Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5′ splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities.     

Solid phase synthesis of somatostatin-28

The synthesis of ovine hypothalamic somatostatin-28 (Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-$\text{Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys}$-OH) has been accomplished by solid phase methodology. The structure of the synthetic material was verified by: (1) direct sequence analysis with a Beckman 89°C sequencer, (2) correlation of the amino acid analyses of the isolated tryptic peptide fragments with their theoretical compositions, and (3) comparison, using high performance liquid chromatography, of the synthetic methionine-sulfoxide and methionine-sulfone modified NH₂-terminal peptides (residues 1–11) with the corresponding tryptic fragment from somatostatin-28.     

Mutations in Either TUBB or MAPRE2 Cause Circumferential Skin Creases Kunze Type

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a β-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect.