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The chlamydiae are obligate intracellular parasites that develop and multiply within a vacuole (termed an inclusion) that does not fuse with lysosomes. Inclusion morphology varies dramatically among the different chlamydiae, particularly within the species Chlamydia psittaci. Some strains develop within a single vacuole, while the mature inclusion of other strains consists of several distinct lobes, each filled with chlamydial developmental forms. The development of this lobed structure was investigated in HeLa cells infected with the guinea pig inclusion conjunctivitis (GPIC) strain of C. psittaci. We employed two recently described probes for the chlamydial inclusion to study the development of these unique lobed structures. The novel probes were an antiserum directed at a protein localized to the GPIC inclusion membrane (anti-IncA) and the fluorescent sphingolipid (N-[7-(4-nitrobenzo-2-oxa-1,3-)]) aminocaproyl sphingosine (NBD-ceramide). Lobed inclusions developed in cells infected at very low multiplicities of infection, suggesting that the structure is not a function of infection by more than one elementary body (EB). Double-label fluorescent-antibody analysis with anti-IncA and an antibody directed at a chlamydial outer membrane protein showed that, prior to 18 h postinfection (p.i.), the inclusion membrane and the chlamydial membrane were tightly associated. After 18 to 20 h p.i., the lobes began to expand and fill with developmental forms and the inclusion membrane and chlamydial membrane became distinct. At times from 8 to 48 h p.i., GPIC inclusions were shown to receive fluorescent derivatives of NBD-ceramide and to be localized to the perinuclear region of the host cell. Labeled lectins with affinity for carbohydrate moieties localized to the Golgi apparatus showed that the lobes of mature inclusions surround the Golgi apparatus. Labeling with NBD-ceramide and the Golgi apparatus-specific lectins therefore demonstrated a functional and physical association of the inclusion with the Golgi apparatus throughout the developmental cycle. Collectively, these results lead to a model for the development of the lobed chlamydial inclusion. We propose that the lobed structure is a result of division of inclusions occurring in parallel with the multiplication of reticulate bodies (RB) early in the developmental cycle. The division of inclusions slows or stops in mid-cycle, and dividing RB accumulate within the enlarging lobes. The RB then differentiate to EBs, the inclusion and cell are lysed, and EBs are freed to infect another cell.
OBJECTIVE: To study the factors affecting cyclodextrin (CD) inclusion of the volatile components in compound traditional Chinese medicines and evaluate the stability of the inclusion compound. METHODS: This study took Gengnian compound as an example to examine the factors affecting the inclusion process according to the inclusion compound utilization ratio. Orthogonal design method was employed optimize the parameters in the inclusion process. The moisture absorption rate of the beta-CD inclusion compound was determined in different humidity and Q10 was used to predict its. RESULTS: The inclusion method, inclusion compound dosage, temperature, inclusion time, and the drying method could all obviously influence the inclusion process. The results of orthogonal design study showed that the optimal beta-CD inclusion of Gengnian compound volatile components could be achieved by adding 8-fold volume of beta-CD and stirring for 3 h at room temperature. Stability experiment showed that the humility did not significantly influence the inclusion compound, which can be stable for 1.26 years. CONCLUSION: This study comprehensively examines the factors affecting the inclusion process and the stability of the inclusion compound, and provides experimental basis for application and study of the inclusion technology.