gD-Independent Superinfection Exclusion of Alphaherpesviruses.

UNLABELLED: Many viruses have the capacity to prevent a cell from being infected by a second virus, often termed superinfection exclusion. Alphaherpesviruses, including the human pathogen herpes simplex virus 1 (HSV-1) and the animal herpesvirus pseudorabies virus (PRV), encode a membrane-bound glycoprotein, gD, that can interfere with subsequent virion entry. We sought to characterize the timing and mechanism of superinfection exclusion during HSV-1 and PRV infection. To this end, we utilized recombinant viruses expressing fluorescent protein (FP) markers of infection that allowed the visualization of viral infections by microscopy and flow cytometry as well as the differentiation of viral progeny. Our results demonstrated the majority of HSV-1- and PRV-infected cells establish superinfection exclusion by 2 h postinfection. The modification of viral infections by virion inactivation and phosphonoacetic acid, cycloheximide, and actinomycin D treatments indicated new protein synthesis is needed to establish superinfection exclusion. Primary infection with gene deletion PRV recombinants identified that new gD expression is not required to establish superinfection exclusion of a secondary viral inoculum. We also identified the timing of coinfection events during axon-to-cell spread, with most occurring within a 2-h window, suggesting a role for cellular superinfection exclusion during neuroinvasive spread of infection. In summary, we have characterized a gD-independent mechanism of superinfection exclusion established by two members of the alphaherpesvirus family and identified a potential role of exclusion during the pathogenic spread of infection. IMPORTANCE: Superinfection exclusion is a widely observed phenomenon initiated by a primary viral infection to prevent further viruses from infecting the same cell. The capacity for alphaherpesviruses to infect the same cell impacts rates of interviral recombination and disease. Interviral recombination allows genome diversification, facilitating the development of resistance to antiviral therapeutics and evasion of vaccine-mediated immune responses. Our results demonstrate superinfection exclusion occurs early, through a gD-independent process, and is important in the directed spread of infection. Identifying when and where in an infected host viral genomes are more likely to coinfect the same cell and generate viral recombinants will enhance the development of effective antiviral therapies and interventions.

On the selective cleavage of nitrous oxide by metal-amide complexes.

Computational investigation of nitrous oxide cleavage by metal-amide systems has shown that a bimetallic mechanism is compatible with the remarkable experimental observation of selective N-N bond scission by a di-molybdenum system, and that the interplay between the bonding energetics of reactants and products can both provide an explanation for the observed cleavage selectivity and be utilized to modify and design chemical behaviour.

Using non-geriatric clinical indicators in a department of rehabilitation and aged care.

We describe an audit using Gastroenterology Clinical Indicators (CIs) to measure quality of care for older patients with gastrointestinal haemorrhage. The gastroenterology CI for gastroscopy within 24 hours of admission was 60%, it was 70% for diagnosis of a cause of acute gastrointestinal bleeding after upper gastroscopy, and it was 30% for death after blood transfusion in a geriatric restorative unit. We discuss whether it is appropriate for a hospital department (Geriatric Medicine) to use the CIs for the specialty (Gastroenterology) providing the service to measure the quality of service being provided. This may be a useful approach given the trend towards cost recouping between different clinical departments.

Paget's disease: acquired resistance to one aminobisphosphonate with retained response to another.

Twenty-five years after the first paper on etidronate in Paget's disease, there are few published papers that address bisphosphonate resistance as a specific clinical phenomenon. We report our data from two studies. Study 1 is a retrospective study of 20 patients with moderate to severe disease who were treated with intravenous (iv) pamidronate (221 +/- 18 mg [SEM]; range 60-360 mg), and after biochemical remission and relapse were retreated with generally larger iv dosage (293 +/- 28 mg; range 180-600 mg). The nadir bone turnover values were similar: plasma alkaline phosphatase (pAP) in 20 patients was 243 +/- 40 IU/l (mean +/- SEM) after the first course, and 267 +/- 44 IU/l after the second (reference range [RR] 35-135 IU/l). Likewise, fasting urinary hydroxyproline excretion (HypE) in 14 of the 20 patients was 4.5 +/- 1.1 micromol/LGF and 4.1 +/- 0.9 micromol/LGF, respectively (RR 0.40-1.92 micromol/LGF). However the minimum duration of biochemical remission was significantly shorter after the second course-10.9 +/- 1.7 months (first) and 5.6 +/- 0.9 months (second) (p < 0.03; Friedman's ANOVA n = 17). A subgroup of 10 patients who were followed for three courses showed a significantly higher pAP nadir in the third course. Study 2 is a prospective study of 40 patients, 23 previously untreated (NILPREV) and 17 previously treated with iv pamidronate (PAMPREV) and in biochemical relapse, who were randomly allocated to either oral alendronate 40 mg daily in 3 month units, or iv pamidronate 60 mg every 3 months. Treatment was continued until pAP and fasting urinary deoxypyridinoline/creatinine (Dpy/Cr) ratios (RR 5-27 micromol/mol) were both in the reference range, or a clear plateau in each marker developed. At baseline, there were no significant differences in either marker between the two NILPREV groups and between the two PAMPREV groups. Using log-transformed data, in NILPREV the pAP reductions were significant and similar over the first 6 months. However, although each Dpy/Cr reduction was also significant, the difference in responses favored alendronate (p < 0.015). In PAMPREV both markers showed no significant response to pamidronate; comparison showed a significantly greater response to alendronate (pAP p < 0.02; Dpy/Cr p < 0.002). Using two-way ANOVA, the pAP responses to alendronate in NILPREV and PAMPREV were similar and those to pamidronate were different (p = 0.034). The percentage of patients with both markers in the RR at 6 months or earlier were identical in NILPREV patients: alendronate 87% and pamidronate 87%. However in PAMPREV they were different: alendronate 83% and pamidronate 0% (p = 0.003). These data indicate: 1) patients treated with the same aminobisphosphonates for two courses show similar nadir values of bone turnover markers but a shorter remission time after the second course. In a third course the nadirs are significantly higher; and 2) in the alendronate/pamidronate comparison, NILPREV and PAMPREV patients showed similar pAP responses to alendronate, but significantly different responses to pamidronate. Thus, patients showing acquired partial resistance to one aminobisphosphonate (usually after two or more previous courses) are still capable of remission after exposure to another compound of the same class.

The effects of calcium supplementation (milk powder or tablets) and exercise on bone density in postmenopausal women.

The etiology of age-related bone loss is unclear but both lack of exercise and dietary calcium deficiency have been implicated in its causation. This 2-year randomized placebo-controlled study was designed to examine the effects of increased dietary calcium and exercise in 168 women who were more than 10 years postmenopausal. The subjects were randomized into one of 4 groups: placebo, milk powder containing 1 g of calcium, calcium tablets 1 g/night, and calcium tablets 1 g/night and an exercise regimen. The exercise group aimed to undertake 4 h of extra weight-bearing exercise per week and were undertaking 10% more activity than other groups at 2 years. Bone mineral density at the lumbar spine, three hip sites, and two sites of the tibia close to the ankle joint were measured at 6 month intervals. Dietary intake was evaluated by a weighed food record, exercise was evaluated by an exercise diary, and blood and urine samples were obtained to examine effects on calcium homeostasis. Individual data points were compared using repeated measures ANOVA and least squares regression. Calcium supplementation by either the calcium tablets or the milk powder resulted in cessation of bone loss at the intertrochanteric hip site (placebo, calcium tablets, calcium and exercise, milk powder -0.81, +0.17, +0.23, and +0.07% per year, respectively; p < 0.05 for all supplementation groups compared with placebo) with similar results at the trochanteric hip site. The calcium and exercise group had less bone loss at the femoral neck site when compared with calcium supplementation alone (placebo, calcium tablets, calcium and exercise, milk powder -0.67, -0.18, +0.28, and -0.18% per year, respectively; p < 0.05 for calcium and exercise compared with calcium alone). There was a significant reduction in the rate of bone loss at the ultradistal site of the tibia (placebo, calcium tablets, calcium and exercise, milk powder -2.5, -1.6, -1.0, and -1.5% per year, respectively; p < 0.05 for all supplementation groups compared with placebo). There was no significant bone loss at the spine site in any group.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of menopause and age on calcitropic hormones: a cross-sectional study of 655 healthy women aged 35 to 90.

Although women lose 30% of their skeletal mass after the menopause, the mechanism of this loss is uncertain. Clearly estrogen deficiency is important but whether this works only through direct effects on the skeleton is uncertain. To examine these mechanisms further we have evaluated calcium-related metabolic factors in 655 healthy women. Fasting blood samples were collected from all subjects who were up to 35 years past the menopause, and fasting urine and 24-h urine samples were collected in 365 women who were up to 25 years past the menopause. In the first 15 years postmenopause, there was a rise in total plasma calcium due to a rise in albumin. Bone resorption (hydroxyproline creatinine ratio), bone formation (alkaline phosphatase), and the urine calcium creatinine ratio all rose at menopause and remained elevated for the next 25 years. There was a transient further rise in bone resorption for the 10 years following menopause. Neither PTH nor the free calcitriol index changed for the first 10 years following menopause. Ten years past the menopause, although total calcitriol rose, the free calcitriol index fell due to a rise in vitamin D binding protein. PTH began to rise at 15 years past menopause. GFR fell gradually over the 25 years following menopause. Thus following menopause there is an increase in bone turnover and increased urine calcium loss independent of any effect of PTH or calcitriol, suggesting a direct effect of estrogen deficiency on bone and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

17 beta-estradiol suppresses gene expression of tartrate-resistant acid phosphatase and carbonic anhydrase II in ovariectomized rats.

Tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II) are key enzymes responsible for osteoclastic bone resorption. In this study, we proposed that estrogen loss in postmenopausal osteoporosis may enhance gene expression of TRACP and CA II, and subsequently increase osteoclastic bone resorption. We have, therefore, used the ovariectomized rat model of postmenopausal bone loss to investigate changes at the gene transcriptional level in osteoclastic bone-resorbing enzymes in ovariectomized (OVX) rats, sham ovariectomized (S-OVX) rats, and estrogen-treated ovariectomized (E-OVX) rats. We have demonstrated for the first time that ovariectomy in rats enhances gene expression of TRACP, and CA II. The mRNA levels in OVX were approximately three- and four-fold higher, respectively, than those in S-OVX. Enhancement was observed 1 week after ovariectomy and transcripts remain high during the experimental period of 8 weeks. Administration of 17 beta-estradiol to OVX (E-OVX) reduced gene expression of these osteoclastic bone-resorbing enzymes 18 hours after injection. It appeared that the suppression of the osteoclastic bone-resorbing enzymes by 17 beta-estradiol was most effective during the first 1-2 weeks but the degree of suppression was reduced at 8 weeks after ovariectomy. In conclusion, our results suggest that estrogen prevents bone loss by reducing the mRNA levels of osteoclastic bone-resorbing enzymes in bone tissue.

The use of actin labelled with N-(1-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin.

A pyrene label attached to Cys-374 of actin has been shown to be a useful probe for monitoring the interaction of actin with myosin subfragments [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38]. We report that the presence of this label decreases the affinity of actin for myosin subfragment 1 by less than a factor of 2. The rate of actin binding is unaffected by the label and the dissociation rate is increased by up to a factor of 2. Both the rate of actin binding to, and the rate of actin dissociation from, heavy meromyosin show two phases when monitored by pyrene fluorescence. Thin filiments reconstituted from pyrene-labelled actin show a 5% increase in pyrene fluorescence on binding Ca2+.

Pressure-relaxation studies of pyrene-labelled actin and myosin subfragment 1 from rabbit skeletal muscle. Evidence for two states of acto-subfragment 1.

We have used actin labelled at Cys-374 with N-(1-pyrenyl)iodoacetamide [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38] to monitor pressure-induced relaxations of acto-myosin subfragment 1. This label greatly increases the sensitivity of measurement of dissociated actin and reveals the presence of two relaxations. The experimental data can be fitted by a model in which actin binds subfragment 1 relatively weakly (K = 5.9 X 10(4) M-1) and then isomerizes to a more tightly bound complex (K = 1.7 X 10(7) M-1). This directly observed isomerization supports the model of Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell. Motil. 5, 351-361]. The rate of the isomerization is too high to be observed in the pressure-jump apparatus (less than 200 microseconds), but analysis of the amplitudes allows estimation of the equilibrium constant of the isomerization as 280 (20 degrees C, 0.1 M-KCl, pH 7). The equilibrium is sensitive to temperature, pressure, ionic strength and the presence of ethylene glycol. The pressure-sensitivity of the isomerization suggests a significant conformational change of the acto-myosin subfragment 1 complex.