Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes

Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At approximately 1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61beta but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61beta- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.     

Case report of pneumatic staple gun injury to the aorta

We describe an 8-year-old boy who had asymptomatic hypercalcemia 4 years after radiotherapy involving the left orbit and lungs. A right parathyroid adenoma was diagnosed, and normocalcemia was achieved after its removal. Routine monitoring of serum calcium and phosphate levels is recommended for children after head and neck irradiation.     

A long follow-up on prostate specific antigen density and prostate biopsy

OBJECTIVE: To determine prostate specific antigen density (PSAD) in a risk population without evidence of prostatic cancer, and to assess the long-term usefulness of PSAD as a parameter for determining the need for a prostatic biopsy in patients with a normal digital rectal examination (DRE) and transrectal ultrasound (TRUS). METHODS: The records of 582 patients referred to the clinic between February, 1992 and February, 1994 were studied retrospectively. All these patients with lower urinary tract symptoms (LUTS) were evaluated based on the following parameters: digital rectal examination, serum PSA levels, prostate volume measured using transrectal ultrasound and PSAD. Prostatic biopsy was performed on 431 patients who had a serum PSA level greater than 4.0 ng/mL. A total of 299 patients (69.3%) had PSA levels between 4.0 and 10.0 ng/mL and represented the target population. The study had two parts, in the first one cancer was diagnosed just by one biopsy and in part II, the patients with negative biopsy in part I were followed for a two-year period and required 2 or 3 biopsies for diagnosis. Of the total of patients who had a negative prostate biopsy in part I of the study, 269 were followed for a period of two years with repeated prostate biopsies. RESULTS: Overall prostate cancer was detected in 22/299 (13.9%) patients, 6/105 (5.7%) with PSAD up to 0.15 and 16/194 (8.2%) with PSAD over 0.15 (p = 0.569). CONCLUSION: PSAD is a useful indicator in decreasing the number of negative biopsies in patients with benign prostatic hyperplasia. However, in a long-term follow-up the PSAD (cutoff level 0.15) was unable to predict which patients had a positive biopsy. According to our results, 5.6% of patients with prostate cancer will be missed using the PSAD criteria.     

Clarification of the relationship between free radical spin trapping and carbon tetrachloride metabolism in microsomal systems

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/.CCl3) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/.CO2-) or the glutathione (GSH) and CCl4-dependent PBN radical adduct (PBN/[GSH-.CCl3]). Inclusion of PBN/.CCl3 in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-.CCl3] or PBN/.CO2- radical adducts. Microsomes alone or with GSH had no effect on the PBN/.CCl3 radical adduct. Addition of NADPH to a microsomal system containing PBN/.CCl3 presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/.CCl3. We conclude that PBN/.CCl3 is not metabolized into either PBN/[GSH-.CCl3] or PBN/.CO2- in microsomal systems.     

Microjet impingement followed by scanning electron microscopy as a qualitative technique to compare cellular adhesion to various biomaterials

Adhesion of cells to biomaterial surfaces is one of the major factors which mediates their biocompatibility. Quantitative or qualitative cell adhesion measurements would be useful for screening new implant materials. Microjet impingement has been evaluated by scanning electron microscopy, to determine to what extent it measures cell adhesion. The shear forces of the impingement, on the materials tested here, are seen to be greater than the cohesive strength of the cells in the impinged area, causing their rupture. The cell bodies are removed during impingement, leaving the sites of adhesion and other cellular material behind. Thus the method is shown not to provide quantification of cell adhesion forces for the metals and culture plastic tested. It is suggested that with highly adherent biomaterials, the distribution and patterns of these adhesion sites could be used for qualitative comparisons for screening of implant surfaces.     

Adrenocorticotropic hormone activation of adenylate cyclase in raphe neurons: multiple regulatory pathways control serotonergic neuronal differentiation

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen (tsT-ag). The RN46A cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature. Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) but no detectable levels of serotonin (5-HT). Treatment of cultures with the adrenocorticotropic hormone peptide ACTH4-10 up-regulates the expression of TPH immunoreactivity in differentiated RN46A cells, but 5-HT synthesis requires initial treatment with ACTH4-10, followed by partial membrane depolarizing conditions. Up-regulation of TPH by ACTH4-10 is apparently due to activation of adenylate cyclase, whereas the increased 5-HT synthesis with membrane depolarization can be blocked with the voltage-sensitive Ca(2+)-channel blockers nifedipine and omega-conotoxin. ACTH4-10 treatment also markedly up-regulates the expression of the 5-HT reuptake transporter, as do dibutyryl cyclic AMP and forskolin; chronic membrane depolarization has no effect on 5-HT reuptake. The expression of the high-affinity 5-HT1A receptor is increased threefold by ACTH4-10 treatment during differentiation and fivefold by differentiation under partial membrane depolarizing conditions. Combining ACTH4-10 treatment and membrane depolarization does not increase expression of the 5-HT1A receptor further. 5-HT release is constitutive in ACTH-treated RN46A cells and linked to spontaneous synaptic vesicle fusion in RN46A cells. Considered with previous results, these data indicate that multiple effectors, ACTH, brain-derived neurotrophic factor, and membrane depolarization, have both distinct and overlapping effects that regulate specific elements of the serotonergic neuronal phenotype during differentiation and maturation.