A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen

Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR.     

Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells

Mammalian cells possess two distinct pathways for completion of base excision repair (BER): the DNA polymerase beta (Pol beta)-dependent short-patch pathway (replacement of one nucleotide), which is the main route, and the long-patch pathway (resynthesis of 2-6 nucleotides), which is PCNA-dependent. To address the issue of how these two pathways share their role in BER the ability of Pol beta-defective mammalian cell extracts to repair a single abasic site constructed in a circular duplex plasmid molecule was tested in a standard in vitro repair reaction. Pol beta-deficient extracts were able to perform both BER pathways. However, in the case of the short-patch BER, the repair kinetics was significantly slower than with Pol beta-proficient extracts, while the efficiency of the long-patch synthesis was unaffected by the loss of Pol beta. The repair synthesis was fully dependent on PCNA for the replacement of long patches. These data give the first evidence that in cell extracts DNA polymerases other than Pol beta are specifically involved in the long-patch BER. These DNA polymerases are also able to perform short-patch BER in the absence of PCNA, although less efficiently than Pol beta. These findings lead to a novel model whereby the two BER pathways are characterized by different protein requirements, and a functional redundancy at the level of DNA polymerases provides cells with backup systems.     

Clinical and in situ cellular responses to Haemophilus ducreyi in the presence or absence of HIV infection

This study examined the prevalence of cardiovascular risk factors among low-income women and assessed the level of awareness and attitudes about these risk factors in the community. A survey instrument was developed and administered by a single researcher to a convenience sample of women in health clinics and nonclinical community settings. These settings included: an academic clinic, community clinics, women's shelters, free meal sites, community centers, public housing units, and private homes in Philadelphia, Pennsylvania. Two hundred two women were selected without regard to age or race. The mean number of cardiovascular risk factors per subject was 2.6 (SD 1.4). Each of eight established cardiovascular risk factors was identified by 4% to 34% of subjects. Among those women with a specific risk factor, only 0% to 45% reported that they were at increased risk due to the presence of that factor. The prevalence of cardiovascular risk factors among low-income women is substantial. Knowledge and understanding of these risk factors is suboptimal, particularly among women personally affected by risk factors for cardiovascular disease.     

Temporal relationship modeling: DTP or DT immunizations and infantile spasms

The time relationship between DTP immunization and infantile spasms (IS) onset was examined using three models--association, temporal shift, and no-effect--and the case/control data from the National Childhood Encephalopathy Study (NCES). Infantile spasms cases classified as being previously abnormal (e.g., tuberous sclerosis complex patients) showed a no-effect relationship, whereas those classified as previously normal suggested a fit to the temporal shift model, i.e. no increase in number of cases but a shortening of time to onset of seizure. No data fit the association model. Analyses for vaccine complications should examine for temporal changes (i.e. temporal shift) in addition to increased risks.     

Sulfur reduction by human erythrocytes

Washed human erythrocytes incubated with glucose and S8 and purged with N2 produced H2S at a nearly constant rate of 170 mumol (L cells)-1 min-1, which continued for several hours. In sealed vials up to 25 mM HS- accumulated. Glucose caused the fastest H2S production, although either lactate or glycerol could support slower rates. When glucose was added without S8, anoxic H2S production nonetheless occurred at about 1.5% of the maximum rate, after 24 hr totaling 0.5 mmol H2S (L cells)-1, suggesting the presence of endogenous reducible sulfur. Anaerobic conditions were not required, since oxygenated cells produced H2S from S8 at 80% of the anoxic rate. Using cell lysates, production of H2S occurred after addition of either glutathione, NADH, or NADPH. The observations suggest possible physiological roles for H2S as an electron carrier, and are consistent with an evolutionary relationship between eukaryotic cytoplasm and sulfur-reducing Archaea.     

Potential roles of conserved amino acids in the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase

The known mammalian 3':5'-cyclic nucleotide phosphodiesterases (PDEs) contain a conserved region located toward the carboxyl terminus, which constitutes a catalytic domain. To identify amino acids that are important for catalysis, we introduced substitutions at 23 conserved residues within the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE; PDE5). Wild-type and mutant proteins were compared with respect to Km for cGMP, kcat, and IC50 for zaprinast. The most dramatic decrease in kcat was seen with H643A and D754A mutants with the decrease in free energy of binding (DeltaDeltaGT) being about 4.5 kcal/mol for each, which is within the range predicted for loss of a hydrogen bond involving a charged residue. His643 and Asp754 are conserved in all known PDEs and are strong candidates to be directly involved in catalysis. Substitutions of His603, His607, His647, Glu672, and Asp714 also produced marked changes in kcat, and these residues are likely to be important for efficient catalysis. The Y602A and E775A mutants exhibited the most dramatic increases in Km for cGMP, with calculated DeltaDeltaGT of 2.9 and 2.8 kcal/mol, respectively, that these two residues are important for cGMP binding in the catalytic site. Zaprinast is a potent competitive inhibitor of cGB-PDE, but the key residues for its binding differ significantly from those that bind cGMP.     

Expression of CD54 (intercellular adhesion molecule-1) and the beta 1 integrin CD29 is modulated by a cyclic AMP dependent pathway in activated primary rat microglial cell cultures

Integrins mediate cell attachment to a variety of extracellular matrix proteins. These interactions play an important role in morphogenesis and differentiation. The mediating functions of integrins during chondrogenesis in vitro were investigated by using mesenchymal cells from limb buds of day 12 mouse embryos. The cells were treated with anti-beta 1, -alpha 1, and -alpha 5 integrin antibodies (a) from day 1 to day 3 and (b) from day 3 to day 7 of cultivation. The total culture period was 7 days. The presence of exogenous anti-beta 1, but not -alpha 1 and -alpha 5 integrin antibodies, from day 1 to 3 completely inhibited the differentiation of blastemal cells to chondroblasts and the formation of cartilage matrix. On the other hand, the presence of exogenous anti-beta 1, -alpha 1, and -alpha 5 integrin antibodies from day 3 of cultivation onwards had no effect. Immunoblotting and immunomorphological findings in the cultures treated with anti-beta 1 antibody from day 1 to day 3 revealed a pattern of integrins and collagen composed of beta 1, alpha 1, alpha 5 beta 1 integrins and collagen type I. The cartilage-specific chondroitin sulfate proteoglycan (CSPG) could not be demonstrated in these cultures. The cultures treated later (day 3 to day 7) showed a pattern of beta 1, alpha 3, alpha 5 beta 1, and alpha v beta 3 integrins, collagen types I and II, and CSPG identical to that of the untreated controls. These findings indicate that beta 1-integrins play a crucial role in early cartilage differentiation and point to a possible important cell-matrix interaction in the induction of chondrogenesis.     

Periodontal repair in intrabony defects treated with a calcium sulfate implant and calcium sulfate barrier

THIS RANDOMIZED, CONTROLLED, CLINICAL STUDY was designed to evaluate outcome following surgical implantation of an allogeneic, freeze-dried, demineralized bone matrix-calcium sulfate (DBM+CS) composite with a CS barrier in intrabony periodontal defects. Twenty-six patients contributing 26 deep intrabony defects completed the study. Thirteen patients received the DBM+CS implant. Thirteen patients received gingival flap surgery alone (GFS; control). Clinical outcome was assessed at 6 and 12 months postsurgery. At 12 months postsurgery, probing depth (PD) reduction (mean +/-SD) for the DBM+CS and GFS group was to 4.3+/-0.5 and 3.0+/-1.3 mm; clinical attachment gain was to 2.9+/-0.8 and 1.7+/-1.5 mm; and probing bone level gain was to 2.9+/-1.4 and 1.2+/-1.2 mm, respectively. There were no apparent differences between evaluations at 6 and 12 months postsurgery. Clinical improvements were significantly different from presurgery for both groups at both observation intervals (P < 0.01). There were no significant differences between groups in PD reduction and clinical attachment gain. Probing bone level gain was significantly greater in the DBM+CS group compared to controls (P < 0.05). In summary, surgical implantation of DBM+CS with a CS barrier resulted in reduced PD and improved attachment levels comparable to that achieved by gingival flap surgery alone. However, gain in probing bone levels in deep intrabony periodontal pockets assessed by clinical parameters was greater than that observed by gingival flap surgery alone. These changes were noted at both 6 and 12 months after surgery. This regenerative technique needs further biologic evaluation before being generally accepted.     

Cartilage changes caused by a coronal surface stepoff in a rabbit model

Coronal stepoffs of 0.5 mm (equal to the cartilage height) were created on the medial femoral condyles of adult, skeletally mature rabbits as a model for articular surface incongruity. After 3, 6, 12, and 24 weeks, tissue was analyzed histologically using hematoxylin and eosin and Safranin O staining, autoradiographs were made of the femoral condyles, and immunohistologic analysis was done for 3-B-3(-) and 7-D-4 chondroitin sulfate epitopes. An overlapping flap from the high toward the low side and an increase of the cartilage height on the low side of the defect were observed as permanent features of adaptation throughout the entire followup. Significant degeneration was not seen around the lesion or in the tibial cartilage opposing a stepoff defect. Autoradiography showed a three-phase response to the lesion: an early increase in radiolabeled sulfate (35SO4) uptake, a sharp decline of 35SO4 uptake, and finally a late recovery of the autoradiographic signal indicating partial recovery of proteoglycan synthetic activity. After an early increase, immunohistologic analysis for 3-B-3(-) showed a subsiding tendency by 24 weeks, and the staining with 7-D-4 remained elevated uniformly in the vicinity of the lesion. A rabbit femoral stepoff defect with an offset of 0.5 mm may remodel and not lead to degeneration within the first 6 months after injury in a stable joint.