Relating cistrons and functions in bacteriophage PM2

An approach to correlate functions with cistrons in bacteriophage PM2 is presented. Two of the temperature-sensitive mutants obtained differed from the wild type in heat sensitivity and four differed in host range. Comparison of the electrophoretic patterns of DNA from cells infected at the restrictive temperature with those obtained in wildtype infection revealed that viral DNA bands were absent with three additional mutants. Complementation analysis assigned the four host range mutants to cistron I and the three mutants defective in DNA synthesis to cistron IV. Recombination frequencies were used to locate cistrons III and IV on the partial genetic map of PM2.     

Reactivity of microhemagglutination, fluorescent treponemal antibody absorption, Venereal Disease Research Laboratory, and rapid plasma reagin tests in primary syphilis.

Seroreactivity of sera from 109 patients with first-infection primary syphilis was 98.2% in the fluorescent treponemal antibody absorption test, 92.7% in the rapid plasma reagin 18-mm circle card test, 72.5% in the microhemagglutination test (MHA-TP), and 72.5% in the Venereal Disease Research Laboratory test. Seroreactivity of sera from 18 patients with primary syphilis with documented previous infection(s) was 100% in the fluorescent treponemal antibody absorption test, the rapid plasma reagin 18-mm circle card test, and the MHA-TP test and 88.9% in the Venereal Disease Research Laboratory test. The MHA-TP test failed to confirm reactivity in 13 of 79 sera which were reactive in the Venereal Disease Research Laboratory test and in 24 of 101 sera which were reactive in the rapid plasma reagin 18-mm circle card test. Testing another production lot of MHA-TP reagents resulted in even poorer correlation. The reactivity of the MHA-TP test in primary syphilis appeared to vary with the sensitivity of the production lot of reagents.     

T cell developmental defects in 'viable motheaten' mice deficient in SHP-1 protein-tyrosine phosphatase. Developmental defects are corrected in vitro in the presence of normal hematopoietic-origin stromal cells and in vivo by exogenous IL-7

Defects in the gene that encodes SHP-1 protein tyrosine phosphatase result in multiple hematopoietic abnormalities and generalized autoimmunity in viable motheaten (me(v)) mice. These mice also exhibit early thymic involution and abnormalities in T cell development. Here, we describe the use of fetal thymic organ culture (FTOC) and bone marrow adoptive transfer to study the effects of SHP-1 deficiency on thymocyte development. Chimeric FTOC established with normal bone marrow placed onto deoxyguanosine-treated fetal thymic lobes or onto scid fetal thymic lobes generated T cells. Bone marrow from SHP-1-deficient me(v)/ me(v) mice generated decreased numbers of T cells in chimeric FTOC established using deoxyguanosine-treated thymi but generated normal numbers in chimeric FTOC established using scid thymi. However, scid fetal thymi seeded with me(v)/ me(v) bone marrow also exhibited morphological abnormalities and contained elevated numbers of macrophages. Addition of IL-7 to me(v)/ me(v) bone marrow-seeded scid FTOC led to increased cell numbers, particularly of macrophages. Intrathymic injection of IL-7 partially restored the ability of progenitor cells in me(v)/ me(v) bone marrow to populate the thymus of adoptive recipients. We conclude that abnormal T cell development in me(v)/ me(v) mice may in part be due to defects in the ability of bone marrow-derived accessory cells to provide bioavailable IL-7 to developing thymocytes.     

Sodium depletion of pregnant ewes and its effects on foetuses and foetal fluids

1. Some effects of sodium depletion were investigated in sheep at different stages of pregnancy ranging from 55 to 139 days. Sodium depletion was induced by draining the saliva from one parotid gland for a period of 6 days. Foetal samples were collected at the end of the depletion period.2. Sodium depletion of the ewe resulted in a fall in saliva Na(+) level and a rise in saliva K(+) level, a fall in plasma Na(+) and K(+) levels, a 35% reduction in plasma volume and a 16% reduction in body weight.3. In the Na(+) depleted ewes the sodium levels of the foetal plasma and amniotic fluid were lower and the volume of the allantoic fluid greater than those in the control ewes.4. It is concluded that sodium depletion of the ewe leads to a deficiency of sodium in the foetus.5. On the basis of these experiments and other reports it is postulated that a sodium deficient foetus, like a sodium deficient adult, responds to the deficiency by restricting sodium losses in the urine and by excreting water.     

Potential modifier role of the R618Q variant of proalpha2(I)collagen in type I collagen fibrillogenesis: in vitro assembly analysis

An arginine to glutamine substitution in the triple helix of proalpha2(I)collagen (R618Q) was first reported in a patient with a variant of Marfan syndrome and later identified in conjunction with a second mutation in a patient with osteogenesis imperfecta (OI). The presence of the R618Q proalpha2(I)collagen allele in unaffected or mildly affected family members suggests that the R618Q allele is either a non-affecting polymorphism or a potential genetic modifier. Conservation of arginine618 across species and fibrillar collagen types suggests it is functionally significant. To investigate the functional significance of the R618Q proalpha2(I)collagen allele, we isolated type I collagen from cultured dermal fibroblasts of control and two unrelated individuals heterozygous for the R618Q proalpha2(I)collagen allele and evaluated helical stability and fibrillar assembly. Type I collagen thermal stability analyzed by protease susceptibility and CD spectroscopy demonstrated no statistical difference between control and R618Q containing collagen molecules. In vitro fibril assembly analyses demonstrated that R618Q containing collagen exhibits rapid fibrillar growth with minimal fibril nucleation phase. Further, electron microscopy demonstrated that the diameter of assembled R618Q containing collagen fibrils was approximately 20% of control collagen fibrils. These findings suggest the R618Q variant does not impact triple helical stability but has a role in collagen fibril assembly, supporting the hypothesis that the R618Q proalpha2(I)collagen variant is a modifier of connective tissue structure/function and is potentially involved in disease pathogenesis.     

Neuronal histamine in the gut wall releasable by gastrin and cholecystokinin

Histamine accumulated in the ligated vagus nerve of the rat, both above and below the ligature; maximum accumulation was after 4 h. The finding is suggestive of axonal flow. Further evidence for histamine in peripheral nerves was obtained in experiments showing that the guinea-pig gut wall could be labelled with [3H]histamine. The experiments were carried out with isolated strips of stomach wall and taenia coli. Electrical stimulation released [3H]histamine from these specimens. The release could be blocked by Ca2+-free medium or by tetrodotoxin. The release was unaffected by vagal denervation or chemical sympathectomy (6-hydroxydopamine) but prevented by reserpinization. Gastrin-17 and cholecystokinin-39 released radioactivity by a tetrodotoxin-sensitive mechanism. The possible existence of a gastrin/cholecystokinin-sensitive neuronal pool of histamine in the gut wall offers a new perspective on the postulated role of histamine as a physiological stimulant of gastric acid secretion and might explain why H2-receptor antagonists block gastrin-stimulated acid secretion.     

Appraisal in the diagnostic laboratory of three commercially available anaerobic cabinets

Three commercially available anaerobic cabinets are described and their performance in relation to one another and to a standard anaerobic jar technique are reported upon from a clinical laboratory.     

Intimin from enteropathogenic Escherichia coli restores murine virulence to a Citrobacter rodentium eaeA mutant: induction of an immunoglobulin A response to intimin and EspB.

The formation of attaching and effacing (A/E) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium (formerly C. freundii biotype 4280)-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the A/E lesion by both EPEC (Int(EPEC)) and C. rodentium (Int(CR)). A secreted protein, EspB (formally EaeB), is also necessary for A/E-lesion formation. Here we report that expression of a cloned Int(EPEC), encoded by plasmid pCVD438, restores murine virulence to an intimin-deficient mutant of C. rodentium DBS255. Replacement of Cys937 with Ala abolished the ability of the cloned EPEC intimin to complement the deletion mutation in DBS255. Ultrastructural examination of tissues from wild-type C. rodentium and DBS255(pCVD438)-infected mice revealed multiple A/E lesion on infected cells and loss of contact between enterocytes and basement membrane. Histological investigation showed that although both wild-type C. rodentium and DBS255(pCVD438) colonized the descending colon and induced colonic hyperplasia in orally infected 21-day-old mice, the latter strain adhered to epithelial cells located deeper within crypts. Nonetheless, infection with the wild-type strain was consistently more virulent, as indicated by a higher mortality rate. All the surviving mice, challenged with either wild-type C. rodentium or DBS255(pCVD438), developed a mucosal immunoglobulin A response to intimin and EspB. These results show that C. rodentium infection provides a relevant, simple, and economic model to investigate the role of EPEC proteins in the formation of A/E lesions in vivo and in intestinal disease.