Phagolysosomes of Coxiella burnetii-infected cell lines maintain an acidic pH during persistent infection.

Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that multiples within vacuoles of phagolysosomal origin. Persistently infected cell lines were maintained in continuous culture for months. We studied the pH of the phagolysosomes by using two murine cell lines during early propagation of the bacteria and after establishment of persistent infection. Three strains of C. burnetii were studied because of the purported propensity of each strain to cause acute or chronic disease and to be resistant or susceptible to antibiotics. The pHs were calculated from fluorescence experiments with fluoresceinated dextran as a lysosomal probe. Phagolysosomal vacuoles maintained an acidic pH during a 36-day infection. Minimal variation of the pH occurred over the duration of the experiment with strains that caused either acute or chronic disease. Phagolysosomal pH remained stable for as long as 153 days with the Nine Mile phase II isolate. Thus, neither the course of C. burnetti infection nor the diversity of antibiotic susceptibility of the strains is related to variations in the phagolysosomal pH.     

Cyclic AMP increases the release of parathyroid hormone-related protein from a lung-cancer cell line

Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of malignant hypercalcemia by stimulating bone resorption and/or renal tubular reabsorption of calcium. In cultured cancer cells, its production can be influenced by various factors or ions, but the regulation of its production is still poorly understood. We investigated the effects of stimulators of cAMP synthesis on PTHrP release by a human lung squamous-carcinoma cell line (BEN). In supervised cells grown on microcarrier beads, PTHrP production was significantly increased after incubation with calcitonin for only 20 min. The release of immunoreactive and bioactive PTHrP was increased by incubating the cells with forskolin, 3-isobutyl-I-methylxanthine or dibutyryl cAMP even in the presence of the protein-synthesis inhibitor cycloheximide for 6 hr. The calcitonin-mediated stimulation was not accompanied by. concomitant changes in PTHrP mRNA. The microfilament-disrupter cytocha-lasin D was shown to enhance the basal and calcitonin-induced production of PTHrP. These results indicate that stimulators of cAMP synthesis enhanced PTHrP release by BEN cells.     

Construction and validation of a Parkinson's disease mutation genotyping array for the Parkin gene.

Parkin mutations account for the majority of familial and sporadic early onset Parkinson's disease (EOPD) cases with a known genetic association. More than 100 mutations have been described in the Parkin gene that includes homozygous, compound heterozygous, and single heterozygous mutations. We have designed a Parkin mutation genotyping array (gene chip) that includes published Parkin sequence variants and allows their simultaneous detection. The chip was validated by screening 85 PD cases and 47 controls previously tested for Parkin mutations. Similar genotyping microarrays have been developed for other genetically heterogeneous diseases including age-related macular degeneration. Here, we show the utility of a genotyping array for Parkinson's disease by analysis of 60 subjects from the Genetic Epidemiology of Parkinson Disease (GEPD) study that includes 15 early-onset PD case probands and 45 relatives.     

Peripheral avian yolk assemblage and its persistence in the blastoderm, studied by trypan blue-induced fluorescence

Summary Shortly after subcutaneous or intraperitoneal injection of nontoxic quantities of trypan blue into laying Japanese quails, red fluorescent yolk granules appear in the peripheral ooplasm of their oocytes at the end of the lampbrush stage or subsequently. Later a red fluorescence can be observed in the apical cytoplasm of the granulosa cells. The results obtained by this method confirm our previous results (Callebaut 1974) obtained by autoradiography after ³H-leucine administration and furnish interesting additional data. The trypan blue-induced fluorescence method gives a good indication of the permeability of the oocytal cortex and its derivative the germinal disc. The avian yolk which is, or has been peripherally assembled (primordial, true white and yellow yolk) can be characteristically labelled by the administration of trypan blue. The injection of higher, still nontoxic quantities of trypan blue has a prolonged retarding effect and permits the marking of a broader part of the germinal disc or eventually of the blastoderm which develops from it.     

Bartonella (Rochalimaea) quintana infections.

Bartonella (formerly Rochalimaea) quintana is the etiological agent of trench fever, a disease extensively reported during the World Wars. Recent molecular biology approaches have allowed dramatic extension of the spectrum of Bartonella infections. B. quintana is now also recognized as an etiological agent of fever and bacteremia, endocarditis, bacillary angiomatosis, and chronic lymphadenopathy. Human immunodeficiency virus-infected patients and/or homeless people are the most vulnerable to infection. Poverty and louse infestation were the main epidemiological factors associated with B. quintana infections during wartime. Although poverty and chronic alcoholism have been associated with modern cases of trench fever and bacteremia due to B. quintana in Europe and the United States, vectors for B. quintana have not been clearly identified and B. quintana has not been isolated from modern-day lice. Microscopic bacillary angiomatosis lesions are characterized by tumor-like capillary lobules, with proliferating endothelial cells. In vitro experiments have shown that B. quintana survives within endothelial cells and stimulates cell proliferation. These observations, together with the finding that lesions may regress when antibiotic therapy is administered, strongly suggest that B. quintana itself stimulates angiogenesis. Bartonella infections are characterized by a high frequency of relapses after brief courses of antibiotic therapy. It is to be noted that in vitro, although Bartonella species are highly susceptible to antibiotics, only the aminoglycosides have proved to be bactericidal. However, the most effective antibiotic regimen for Bartonella infections remains to be established.     

Clinical evidence of the nonpathogenic nature of the M34T variant in the connexin 26 gene

Mutations in GJB2 are the most common cause of congenital nonsyndromic hearing loss. The controversial allele variant M34T has been hypothesized to cause autosomal dominant or recessive nonsyndromic hearing impairment and some in vitro data has been consistent with this hypothesis. In this report, we present the clinical and genotypic study of 11 families (seven familial forms of nonsyndromic sensorineural hearing loss (NSSNHL) and four sporadic cases) in which the M34T GJB2 variant has been identified. The M34T mutation did not segregate with the deafness in six of the seven familial forms of NSSNH. Eight persons with normal audiogram presented a heterozygous M34T variation and five normal hearing individuals were composite heterozygous for M34T and another GJB2 mutation. Four normal hearing individuals with a documented audiogram were M34T/35delG and one was M34T/(GJB6-D13S1830)del. Screening a French control population of 116 subjects we have found an M34T allele frequency of 1.72%. This percentage was not significatively different from the prevalence of the M34T allele in the deaf population, which was 2.12%. All these data suggest that the M34T variant is not clinically significant in human and is a frequent polymorphism in France.     

A new form of skeletal dysplasia with amelogenesis imperfecta and platyspondyly

We report two patients, born of consanguineous parents, affected by a disorder resulting in mild growth retardation. Hallmarks are amelogenesis imperfecta (absence of the enamel cap) associated with brachyolmia-like anomalies: platyspondyly with short pedicles, narrow intervertebral and interpedicular distances, rectangular-shaped vertebrae with posterior scalloping and herniation of the nuclei, and broad femoral necks. Inheritance appears to be autosomal recessive.     

Pseudoaminopterin syndrome

We report on a boy with a combination of manifestations reminiscent of aminopterin embryopathy: brachyturricephaly with craniosynostosis, poorly mineralised vault, upslanted palpebral fissures, malar hypoplasia, high-arched palate, micrognathia, thick, abnormal auricles, ASD, minor hand anomalies, growth and mental ratardation. Three convincing cases of “Aminopterin Syndrome Sine Aminopterin” have been reported (the fourth case possibly having the Juberg–Hayward syndrome). Variability and heterogeneity of cases with apparent aminopterin embryopathy are discussed. © 1993 Wiley-Liss, Inc.