Involvement of the Golgi region in the intracellular trafficking of cholera toxin

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)-elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID₅₀ value similar to the LD₅₀ of BFA in Y1 adrenal cells. Binding and internalization of [¹²⁵I]-cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK₁ cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different. © 1993 Wiley-Liss, Inc.     

6-Fluorocholesterol as a growth factor for the yeast mutant GL7

6-Fluorocholesterol supports the growth of the sterol-requiring yeast mutant GL7 albeit less efficiently than cholesterol or ergosterol. When the fluoro analogue is combined with very much smaller amounts of cholesterol, the growth response to the sterol pair is synergistic, i.e., greater than additive. On further addition of trace amounts of ergosterol to the 6-fluorocholesterol-cholesterol pair, an additional synergistic growth response is observed. On 6-fluorocholesterol alone, the growth rate of the yeast mutant is slow initially, but after several transfers of such cells to the same media containing the fluoro analogue, growth improves substantially. When incorporated into artificial membranes, cholesterol and its 6-fluoro analogue have essentially identical effects on membrane fluidity as judged from microviscosity measurements. The contrasting responses of artificial membranes and whole cells to the 6-fluoro analogue of cholesterol might be due to sterol-protein interactions in natural membranes.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

Cone Beam Computed Tomography Assessment of the Maxillary Incisive Canal and Foramen: Considerations of Anatomical Variations When Placing Immediate Implants

IntroductionThe maxillary incisive canal connects the roof of the oral cavity with the floor of nasal cavity and has the incisive and nasal foramina respectively at its two opposite ends. Its close proximity with the anterior incisors affects one’s ability to place immediate implants in ideal position.ObjectiveTo avoid causing complication, variations in their dimensions were studied.ResultsThe mean labiopalatal and mesiodistal measurements of the incisive foramen were 2.80mm and 3.49 mm respectively, while the labiopalatal width of the nasal foramen was 6.06mm. The incisive canal was 16.33mm long and 3.85 mm wide. The anterior maxillary bone has an average thickness of 7.63 mm. The dimensions of the incisive foramen and incisive canal, and anterior maxillary bone thickness demonstrated gender differences with males showing greater values. The anterior maxillary bone thickness was affected by age but this difference was not observed in canal dimensions. The majority of subjects have a funnel shape-like incisive canal with the broader opening located at its superior. They seem to have a longer slanted-curve canal with one channel at its middle portion and a narrower incisive foramen opening than those reported elsewhere.ConclusionsThis study found that gender is an important factor that affected the characteristics of the IC and the amount of bone anterior to it. Male generally had bigger IC and thicker anterior bone. In addition, the anterior maxillary bone thickness was affected by aging, where it becomes thinner with increased age even though the subjects were fully dentate.     

Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair

Meiotic programmed DNA double-strand break (DSB) repair is essential for crossing-over and viable gamete formation and requires removal of Spo11-oligonucleotide complexes from 5′ ends (clipping) and their resection to generate invasive 3′-end single-stranded DNA (resection). Ctp1 (Com1, Sae2, CtIP homolog) acting with the Mre11-Rad50-Nbs1 (MRN) complex is required in both steps. We isolated multiple S. pombe ctp1 mutants deficient in clipping but proficient in resection during meiosis. Remarkably, all of the mutations clustered in or near the conserved CxxC or RHR motif in the C-terminal portion. The mutants tested, like ctp1Δ, were clipping-deficient by both genetic and physical assays­. But, unlike ctp1Δ, these mutants were recombination-proficient for Rec12 (Spo11 homolog)-independent break-repair and resection-proficient by physical assay. We conclude that the intracellular Ctp1 C-terminal portion is essential for clipping, while the N-terminal portion is sufficient for DSB end-resection. This conclusion agrees with purified human CtIP resection and endonuclease activities being independent. Our mutants provide intracellular evidence for separable functions of Ctp1. Some mutations truncate Ctp1 in the same region as one of the CtIP mutations linked to the Seckel and Jawad severe developmental syndromes, suggesting that these syndromes are caused by a lack of clipping at DSB ends that require repair.