Nanoscale three-dimensional imaging of the human myocyte.

The ventricular human myocyte is spatially organized for optimal ATP and Ca(2+) delivery to sarcomeric myosin and ionic pumps during every excitation-contraction cycle. Comprehension of three-dimensional geometry of the tightly packed ultrastructure has been derived from discontinuous two-dimensional images, but has never been precisely reconstructed or analyzed in human myocardium. Using a focused ion beam scanning electron microscope, we created nanoscale resolution serial images to quantify the three-dimensional ultrastructure of a human left ventricular myocyte. Transverse tubules (t-tubule), lipid droplets, A-bands, and mitochondria occupy 1.8, 1.9, 10.8, and 27.9% of the myocyte volume, respectively. The complex t-tubule system has a small tortuosity (1.04±0.01), and is composed of long transverse segments with diameters of 317±24nm and short branches. Our data indicates that lipid droplets located well beneath the sarcolemma are proximal to t-tubules, where 59% (13 of 22) of lipid droplet centroids are within 0.50µm of a t-tubule. This spatial association could have an important implication in the development and treatment of heart failure because it connects two independently known pathophysiological alterations, a substrate switch from fatty acids to glucose and t-tubular derangement.

Pneumococcal vaccination programs and the burden of invasive pneumococcal disease in Ontario, Canada, 1995-2011.

BACKGROUND: In 1995, a publicly funded pneumococcal vaccination program for 23-valent polysaccharide vaccine (PPV23) was introduced in Ontario. Conjugate vaccines were authorized in 2001 (PCV7), 2009 (PCV10) and 2010 (PCV13). METHODS: From 1995-2011, active, population-based surveillance for invasive pneumococcal disease (IPD) was conducted in Metropolitan Toronto and Peel Region, Canada. RESULTS: 6404 IPD cases were included. After PPV23 program implementation in 1995, IPD due to PPV23 strains decreased 49% in older adults prior to PCV7 introduction. Estimated PPV23 efficacy in vaccine eligible adults was 42.2% (95% CI; 28.6-53.2%). IPD incidence due to PCV7 serotypes in children <5 years decreased significantly after PCV7 authorization and before introduction of a publicly funded PCV7 program. Seven years after PCV7 program implementation, the incidence of IPD due to PCV7 serotypes decreased to zero in children and by 88% in adults, however, overall IPD incidence remained unchanged in adults. In 2011, the incidence of IPD was 4.5 per 100,000 in adults aged 15-64 and 19.9 per 100,000 in adults aged over 65 years, with 45 serotypes causing disease. Between 1995 and 2011, the case fatality rate of IPD in adults decreased 2% per year (95% CI, -0.9% to -3.2%). In multivariable analysis, predictors of mortality included older age, chronic conditions, nursing home residence, current smoking, bacteraemia, and illness due to serotypes 3,11A, 19A, and 19F. CONCLUSIONS: While vaccination programs resulted in substantial public health benefits, herd immunity benefits of PCV7 were seen at low pediatric vaccination rates, and the case fatality rate of IPD has decreased, IPD will continue to be a cause of considerable morbidity and mortality in adults.

Autoimmune hepatitis in a Jamaican cohort spanning 40 years.

BACKGROUND: The prevalence and characteristics of autoimmune hepatitis (AIH) in black populations are not well documented. OBJECTIVES: To describe the clinical and laboratory features of AIH in patients presenting at a Jamaican hospital, 1969-2009. METHODS: A retrospective review of hospital records was done and patients were classified by the revised international scoring system. RESULTS: Fifty patients satisfied criteria for diagnosis of AIH type-1 and most presented late at hospital. Almost one third of cases presented with chronic liver disease (32%) while 8% presented with fulminant hepatic failure and 2% with acute liver disease. Jaundice (92%) was the most common presenting feature. Other associated autoimmune diseases (10%) systemic lupus erythematosus and insulin dependent diabetes mellitus, were found. All patients (100%) had abnormal liver biochemical tests, 81% had hypergammaglobulinaemia and 82% AIH associated autoantibodies. The prevalence of autoantibodies increased with age (P=0.05). Liver biopsy, performed in 33 cases, showed chronic hepatitis (45%), cirrhosis (24%) chronic hepatitis with bridging necrosis (15%), chronic hepatitis with rosetting (6%) and nonspecific findings (10%). Patients were treated with prednisone with or without azathioprine. Relapse occurred in 4% and death 6%. CONCLUSION: Autoimmune hepatitis is rare in Jamaicans and prognosis is similar to that reported in developed countries.

Usp14 deficiency increases tau phosphorylation without altering tau degradation or causing tau-dependent deficits.

Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (Usp14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that Usp14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using Usp14-deficient ax(J) mice, we investigated if loss of Usp14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of Usp14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the Usp14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3ß. However, genetic ablation of tau did not alter any of the neurological deficits in the Usp14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of Usp14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.

Effect of insulin and an erythropoietin-derived peptide (ARA290) on established neuritic dystrophy and neuronopathy in Akita (Ins2 Akita) diabetic mouse sympathetic ganglia.

The Akita mouse is a robust model of diabetic autonomic neuropathy which develops severe diabetes following beta cell death, which occurs reproducibly at 3-4 weeks of age, and maintains the diabetic state without therapy for as long as 11 additional months. Neuritic dystrophy and neuronopathy involving prevertebral sympathetic superior mesenteric and celiac ganglia begin to develop within the first two months of onset of diabetes and are progressive with time. We have examined the effect of insulin implants resulting in normoglycemia and injections of ARA290, a small erythropoietin peptide which has no effect on glycemic parameters, on the reversal of established neuritic dystrophy and neuronopathy. We have found that 4 weeks of insulin therapy beginning at 2 months of diabetes resulted in normalization of blood glucose, body weight and HbA1c. Insulin therapy successfully reversed established neuritic dystrophy and neuronopathy to control levels. Numbers of sympathetic neurons were not significantly changed in either 3 month diabetic or insulin-treated Akita mice. Treatment with ARA290 for 7 weeks beginning at 4 months of diabetes did not result in altered metabolic severity of diabetes as measured by blood glucose, body weight or HbA1c levels. ARA290 treatment was able to decrease neuritic dystrophy by 55-74% compared to untreated diabetics or in comparison to a separate group of diabetic animals representing the 4 month treatment onset point. Surprisingly, there was no effect of ARA290 on ganglionic neuron number or ongoing neuronopathy (pale/degenerating neurons) in diabetic Akita mice during this time period. The development of neuroprotective EPO-like peptides may provide a possible future therapy for this debilitating complication of diabetes; however, it appears that discrete elements may be differentially targeted by the diabetic state and may require selective therapy.

Predicting patient discharge disposition after total joint arthroplasty in the United States.

The purpose of this study was to develop an easily administered tool to preoperatively predict patient discharge disposition after total joint arthroplasty in the United States. Data were collected in a retrospective review of 517 medical charts and analyzed using logistic regression to develop a model for predicting the likelihood that a patient will not be discharged directly home. The resulting regression model was the basis for the nomogram, named the Predicting Location after Arthroplasty Nomogram. This model demonstrated a bootstrap-corrected concordance index of 0.867, excellent calibration, and external validation as demonstrated by a concordance index of 0.861. Preoperative knowledge that a patient is likely to require an extended care facility allows the clinical care team to make appropriate arrangements and avoid potential delays in patient discharge.

Population-based surveillance for invasive pneumococcal disease in homeless adults in Toronto.

BACKGROUND: Identification of high-risk populations for serious infection due to S. pneumoniae will permit appropriately targeted prevention programs. METHODS: We conducted prospective, population-based surveillance for invasive pneumococcal disease and laboratory confirmed pneumococcal pneumonia in homeless adults in Toronto, a Canadian city with a total population of 2.5 M, from January 1, 2002 to December 31, 2006. RESULTS: We identified 69 cases of invasive pneumococcal disease and 27 cases of laboratory confirmed pneumococcal pneumonia in an estimated population of 5050 homeless adults. The incidence of invasive pneumococcal disease in homeless adults was 273 infections per 100,000 persons per year, compared to 9 per 100,000 persons per year in the general adult population. Homeless persons with invasive pneumococcal disease were younger than other adults (median age 46 years vs 67 years, P<.001), and more likely than other adults to be smokers (95% vs. 31%, P<.001), to abuse alcohol (62% vs 15%, P<.001), and to use intravenous drugs (42% vs 4%, P<.001). Relative to age matched controls, they were more likely to have underlying lung disease (12/69, 17% vs 17/272, 6%, P = .006), but not more likely to be HIV infected (17/69, 25% vs 58/282, 21%, P = .73). The proportion of patients with recurrent disease was five fold higher for homeless than other adults (7/58, 12% vs. 24/943, 2.5%, P<.001). In homeless adults, 28 (32%) of pneumococcal isolates were of serotypes included in the 7-valent conjugate vaccine, 42 (48%) of serotypes included in the 13-valent conjugate vaccine, and 72 (83%) of serotypes included in the 23-valent polysaccharide vaccine. Although no outbreaks of disease were identified in shelters, there was evidence of clustering of serotypes suggestive of transmission of pathogenic strains within the homeless population. CONCLUSIONS: Homeless persons are at high risk of serious pneumococcal infection. Vaccination, physical structure changes or other program to reduce transmission in shelters, harm reduction programs to reduce rates of smoking, alcohol abuse and infection with bloodborne pathogens, and improved treatment programs for HIV infection may all be effective in reducing the risk.

Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation.

There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1(+/-) mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1(+/-) mice to Becn1(+/-) mice. After 1 week on a high fat diet, 4-week-old Pdx1(+/-) Becn1(+/-) mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1(+/-) mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention.

Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens.

The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. Using mice with granulocyte- and macrophage-specific deletion of the essential autophagy protein Atg5, we show that Atg5 is required for in vivo resistance to the intracellular pathogens Listeria monocytogenes and Toxoplasma gondii. In primary macrophages, Atg5 was required for interferongamma (IFN-gamma)/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect classical hallmarks of autophagy, such as autophagosomes enveloping T. gondii, Atg5 was required for recruitment of IFN-gamma-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane, an event that mediates IFN-gamma-mediated clearance of T. gondii. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo, and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking.

Erythropoietin and its carbamylated derivative prevent the development of experimental diabetic autonomic neuropathy in STZ-induced diabetic NOD-SCID mice.

Autonomic neuropathy is a significant diabetic complication resulting in increased morbidity and mortality. Studies of autopsied diabetic patients and several rodent models demonstrate that the neuropathologic hallmark of diabetic sympathetic autonomic neuropathy in prevertebral ganglia is the occurrence of synaptic pathology resulting in distinctive dystrophic neurites ("neuritic dystrophy"). Our prior studies show that neuritic dystrophy is reversed by exogenous IGF-I administration without altering the metabolic severity of diabetes, i.e. functioning as a neurotrophic substance. The description of erythropoietin (EPO) synergy with IGF-I function and the recent discovery of EPO's multifaceted neuroprotective role suggested it might substitute for IGF-I in treatment of diabetic autonomic neuropathy. Our current studies demonstrate EPO receptor (EPO-R) mRNA in a cDNA set prepared from NGF-maintained rat sympathetic neuron cultures which decreased with NGF deprivation, a result which demonstrates clearly that sympathetic neurons express EPO-R, a result confirmed by immunohistochemistry. Treatment of STZ-diabetic NOD-SCID mice have demonstrated a dramatic preventative effect of EPO and carbamylated EPO (CEPO, which is neuroprotective but not hematopoietic) on the development of neuritic dystrophy. Neither EPO nor CEPO had a demonstrable effect on the metabolic severity of diabetes. Our results coupled with reported salutary effects of EPO on postural hypotension in a few clinical studies of EPO-treated anemic diabetic and non-diabetic patients may reflect a primary neurotrophic effect of EPO on the sympathetic autonomic nervous system, rather than a primary hematopoietic effect. These findings may represent a major clinical advance since EPO has been widely and safely used in anemic patients due to a variety of clinical conditions.

Infection control and antimicrobial restriction practices for antimicrobial-resistant organisms in Canadian tertiary care hospitals.

In 2003, a survey examining infection control and antimicrobial restriction policies and practices for preventing the emergence and transmission of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and extended spectrum beta-lactamase (ESBL) was performed within Canadian teaching hospitals as part of the Canadian Nosocomial Infection Surveillance Program. Twenty-eight of 29 questionnaires were returned. The majority of facilities conducted admission screening for MRSA (96.4%) and VRE (89.3%) but only 1 site screened for ESBL/AmpC. Rates of MRSA, VRE, and ESBL remain low in Canada. It is believed that these lower rates may be due to intense admission screening protocols and stringent infection control policies for antimicrobial-resistant organisms (AROs) within Canadian institutions. Few (MRSA: 14.8%; VRE: 12.0%) recorded the number of patients screened. Regular prevalence surveys were done for MRSA (21.4%), VRE (35.7%), and ESBL/AmpC (3.8%). Pre-emptive precautions were applied for MRSA by 60.7% and for VRE by 75.0% of facilities. All facilities flagged patients previously identified with MRSA and VRE but only 46.2% flagged ESBL and 15.4% flagged AmpC patients. Barrier precautions varied by ARO and patient-care setting. In the inpatient non-ICU setting, more than 90% wore gowns and gloves for MRSA and VRE but only 50% for ESBL; and 57.1% wore masks for MRSA. Attempts to decolonize MRSA patients had been made by 82.1%, largely in order to place them in another facility. Policies restricting antimicrobial prescribing were reported by 21 facilities (75.0%). Further studies examining hospital infection control policies and corresponding rates of ARO infections would help in identifying and refining best practice guidelines within Canadian institutions.

JunD is a profibrogenic transcription factor regulated by Jun N-terminal kinase-independent phosphorylation.

JunD is implicated in the regulation of hepatic stellate cell (HSC) activation and liver fibrosis via its transcriptional regulation of the tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. In the present study we found in vivo evidence of a role for JunD in fibrogenesis. Expression of JunD was demonstrated in alpha-SMA-positive activated HSCs of fibrotic rodents and human livers. The junD-/- mice were protected from carbon tetrachloride-induced fibrosis. The livers of injured junD-/- mice displayed significantly reduced formation of fibrotic crosslinked collagen and a smaller number of alpha-SMA-positive HSCs compared with those of wild-type (wt) mice. Hepatic TIMP-1 mRNA expression in injured junD-/- mice was 78% lower and in culture activated junD-/- HSCs was 50%-80% lower than that in wt mice. In examining the signal transduction mechanisms that regulate JunD-dependent TIMP-1 expression, we found a role for phosphorylation of the Ser100 residue of JunD but ruled out JNK as a mediator of this event, suggesting ERK1/2 is utilized. In conclusion, a signaling pathway for the development of fibrosis involves the regulation of TIMP-1 expression by phosphorylated JunD.

Modeling transmission of methicillin-resistant Staphylococcus aureus among patients admitted to a hospital.

OBJECTIVE: To determine the impact of the screening test, nursing workload, handwashing rates, and dependence of handwashing on risk level of patient visit on methicillin-resistant Staphylococcus aureus (MRSA) transmission among hospitalized patients. SETTING: General medical ward. METHODS: Monte Carlo simulation was used to model MRSA transmission (median rate per 1,000 patient-days). Visits by healthcare workers (HCWs) to patients were simulated, and MRSA was assumed to be transmitted among patients via HCWs. RESULTS: The transmission rate was reduced from 0.89 to 0.56 by the combination of increasing the sensitivity of the screening test from 80% to 99% and being able to report results in 1 day instead of 4 days. Reducing the patient-to-nurse ratio from 4.3 in the day and 6.8 at night to 3.8 and 5.7, respectively, reduced the number of nosocomial infections from 0.89 to 0.85; reducing the ratio to 1 and 1, respectively, further reduced the number of nosocomial infections to 0.32. Increases in handwashing rates by 0%, 10%, and 20% for high-risk visits yielded reductions in nosocomial infections similar to those yielded by increases in handwashing rates for all visits (0.89, 0.36, and 0.24, respectively). Screening all patients for MRSA at admission reduced the transmission rate to 0.81 per 1,000 patient-days from 1.37 if no patients were screened. CONCLUSION: Within the ranges of parameters studied, the most effective strategies for reducing the rate of MRSA transmission were increasing the handwashing rates for visits involving contact with skin or bodily fluid and screening patients for MRSA at admission.

Distinct pathways regulate expression of cardiac electrical and mechanical junction proteins in response to stretch.

To define mechanisms regulating expression of cell-cell junction proteins, we have developed an in vitro system in which neonatal rat ventricular myocytes were subjected to pulsatile stretch. Previously, we showed that expression of the gap junction protein, connexin (Cx) 43, is increased by approximately 2-fold after 1 hour of stretch, and this response is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF). Here, we report that the mechanical junction proteins plakoglobin, desmoplakin, and N-cadherin are also upregulated by pulsatile stretch but by a mechanism independent of VEGF or other secreted chemical signals. Stretch-induced upregulation of mechanical junction proteins was blocked by anti-beta1 and anti-beta3 integrin antibodies. Transfection of cells with adenovirus expressing GFP-FRNK, a dominant-negative inhibitor of focal adhesion kinase (FAK)-dependent signaling, blocked stretch-induced upregulation of Cx43 and mechanical junction proteins but did not block the ability of exogenous VEGF to upregulate Cx43 expression. Conditioned medium removed from uninfected cells after stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretch-conditioned medium from GFP-FRNK cells had no effect on Cx43 expression. The src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine blocked stretch-induced upregulation of mechanical junction proteins but not Cx43. Thus, stretch upregulates expression of both electrical and mechanical junction proteins via integrin-dependent activation of FAK. Stretch-induced upregulation of Cx43 expression is mediated by FAK-dependent secretion of VEGF. In contrast, stretch-induced upregulation of adhesion junction proteins involves intracellular mechanotransduction pathways initiated via integrin signaling and acting downstream of src kinase.

Matrix-protein-specific regulation of Cx43 expression in cardiac myocytes subjected to mechanical load.

To elucidate mechanisms responsible for mechanotransduction in the heart and define the effects of remodeling of the extracellular matrix, we cultured neonatal rat ventricular myocytes on native type I collagen, fibronectin, or denatured collagen and subjected them to uniaxial, pulsatile stretch. Changes in expression of the cardiac gap junction protein, Cx43, were measured by confocal microscopy and immunoblotting. Cells grown on fibronectin or denatured collagen exhibited significantly greater Cx43 expression than cells grown on native collagen. Stretch induced a approximately 2-fold increase in Cx43 expression in cells grown on native collagen but no increase in cells grown on fibronectin or denatured collagen. Incubation of cells on native collagen with a peptide containing the arginine-glycine-aspartate (RGD) motif upregulated Cx43 expression equivalent to that induced by stretch. Nonselective activation of integrin signaling with MnCl2 also upregulated Cx43 expression in cells grown on native collagen. This effect was blocked completely by pretreatment with anti-beta1 integrin antibody but not by anti-beta3 integrin antibody. Stretch led to a marked increase in beta1 integrin immunofluorescent signal in cells grown on native collagen but not in cells grown on fibronectin or denatured collagen. Stretch-induced upregulation of Cx43 was also blocked by anti-beta1 integrin antibody. Thus, matrix protein-myocyte interactions regulate Cx43 expression via beta1 integrin signaling initiated by mechanical stimulation in cells grown on native type I collagen, or by RGD-integrin signaling independent of mechanical stress in cells grown on fibronectin or denatured collagen. Changes in the composition of the extracellular matrix may affect electrical coupling in cardiac myocytes.

Effect of hard-drug use on CD4 cell percentage, HIV RNA level, and progression to AIDS-defining class C events among HIV-infected women.

In vitro and animal studies suggest that cocaine and heroin increase HIV replication and suppress immune function, whereas epidemiologic studies are inconclusive regarding their effect on HIV infection progression. The authors prospectively examined the association between illicit-drug use and 4 outcome measures (CD4 cell percentage, HIV RNA level, survival to class C diagnosis of HIV infection, and death) in a national cohort of HIV-infected women. Women enrolled between 1989 and 1995 were followed for 5 years and repeatedly interviewed about illicit ("hard")--drug use. Up to 3 periodic urine screens validated self-reported use. Outcomes were compared between hard-drug users (women using cocaine, heroin, methadone, or injecting drugs) and nonusers, adjusting for age, antiretroviral therapy, number of pregnancies, smoking, and baseline CD4 cell percentage. Of 1148 women, 40% reported baseline hard-drug use during pregnancy. In multivariate analyses, hard-drug use was not associated with change in CD4 cell percentage (P = 0.84), HIV RNA level (P = 0.48), or all-cause mortality (relative hazard = 1.10; 95% confidence interval, 0.61-1.98). Hard-drug users did, however, exhibit a higher risk of developing class C diagnoses (relative hazard = 1.65; 95% confidence interval, 1.00-2.72), especially herpes, pulmonary tuberculosis, and recurrent pneumonia. Hard-drug-using women may have a higher risk for nonfatal opportunistic infections.

Experimental and structural evidence that herpes 1 kinase and cellular DNA polymerase(s) discriminate on the basis of sugar pucker.

Two isomers of methanocarba (MC) thymidine (T), one an effective antiherpes agent with the pseudosugar moiety locked in the North (N) hemisphere of the pseudorotational cycle (1a, N-MCT) and the other an inactive isomer locked in the antipodean South (S) conformation (1b, S-MCT) were used to determine whether kinases and polymerases discriminate between their substrates on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono-, di-, and triphosphate levels in control cells, cells infected with the Herpes simplex virus, or cells transfected with the corresponding viral kinase gene (HSV-tk), suggests that kinases prefer substrates that adopt the S sugar conformation. On the other hand, the cellular DNA polymerase(s) of a murine tumor cell line transfected with HSV-tk incorporated almost exclusively the triphosphate of the locked N conformer (N-MCTTP), notwithstanding the presence of higher triphosphate levels of the S-conformer (S-MCTTP).

Risk factors for Kaposi's sarcoma-associated herpesvirus infection among HIV-1-infected pregnant women in the USA.

OBJECTIVES: We sought to identify risk factors for infection with the Kaposi's Sarcoma-associated herpesvirus (KSHV) among pregnant women and to examine a reported association of KSHV with injecting drug use (IDU) and hepatitis C virus (HCV) infection. DESIGN: Cross-sectional evaluation of questionnaire data and KSHV and HCV seroprevalence in the Women and Infants Transmission Study. METHODS: In sera collected from HIV-1-infected pregnant women (n = 887) and, at age 12 months, their offspring (n = 900) at six sites in the USA and Puerto Rico, KSHV and HCV antibodies were detected with sensitive and specific enzyme immunoassays. Risk of KSHV was estimated by the unadjusted and adjusted odds ratio (OR(adj)) and 95% confidence interval (CI). The geographic referent sites were Chicago and Boston. RESULTS: Forty-seven (5.3%) of the women and three (0.3%) of the infants were KSHV seropositive. In univariate and multivariate analyses, KSHV in the women was associated with enrollment in Puerto Rico, Houston or Brooklyn (OR(adj), 4.3; 95% CI, 1.8-10.4) or Manhattan (OR(adj), 9.8; 95% CI, 3.7-25.6); non-completion of high school (OR(adj), 1.8; 95% CI, 0.9-3.4); the number of sexually transmitted diseases (OR(adj), 1.4; 95% CI, 1.0-1.9 per disease); and especially with IDU and HCV infection (OR(adj), 3.5; 95% CI, 1.5-7.9). CONCLUSIONS: Transmission of KSHV by blood inoculation may be highly inefficient, but our data support the hypothesis that it does occur. Large formal studies to evaluate whether KSHV transmission occurs via transfusion are needed to inform decisions regarding screening volunteer blood donors to protect the blood supply.

Severe group a streptococcal soft-tissue infections in Ontario: 1992-1996.

A prospective, population-based, surveillance study of invasive soft-tissue infections due to group A streptococci was conducted in Ontario, Canada, from 1992 through 1996. Demographic and clinical information was obtained by patient interview and chart review. Isolates were characterized by M protein and T agglutination typing. The incidence of necrotizing fasciitis (NF) increased from 0.08 cases per 100,000 population in 1992 to 0.49 cases per 100,000 population in 1995. The case-fatality rate was 13% (68 of 520 patients died). Hypotension and multiorgan dysfunction complicated 64 cases (12%), and NF complicated 119 cases (23%). Underlying diabetes, alcohol abuse, cancer, and cardiac and pulmonary disease increased the risk of disease. Prior use of nonsteroidal anti-inflammatory agents did not influence disease severity. All 197 patients without NF, underlying illness, and hypotension at presentation survived, as did 95 (99%) of 96 normotensive patients who were <65 years old but who had underlying chronic illness. Previously healthy patients without hypotension or NF may be considered for outpatient treatment.

Cloning, characterization and functional expression of a phospholipase Dalpha cDNA from tomato fruit.

Phospholipase D (PLD; EC 3.1.4.4) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Phosphatidic acid (PA), the product of PLD hydrolysis of PL, increases in pericarp tissue during ripening of tomato (Lycopersicon esculentum Mill.) fruit, suggesting that increased PLD activity may be involved in loss of membrane function associated with ripening. However, a recent report showed a decline in soluble PLD activity in both normal and nonripening mutant fruit over the span that encompasses full ripening. To directly assess the role of PLD in tomato ripening, we have initiated a molecular genetic approach. Using a PLDalpha cDNA from castor bean as a probe, a PLDalpha cDNA (LEPLD2) was isolated from a tomato fruit library. It has an open reading frame of 2 421 nucleotides, predicted to encode a polypeptide of 807 amino acids, with a molecular mass of 91.9 kDa. These values are close to those of PLDalphas from 11 plant species and LEPLD2 has >/=73% nucleotide sequence identity with PLDalpha cDNAs from castor bean and tobacco, as well as another tomato cDNA. LEPLD2 transcript was detected in all tissues of the tomato plant by RNA gel-blot analysis. Levels were very low in roots, low in stems, moderate in leaves, high in flowers and increased in fruit during development and ripening. Expression of LEPLD2 in Escherichia coli yielded phosphatidylcholine-hydrolyzing enzyme, and cells transformed with a pFLAG-MAC vector construct produced a FLAG-PLDalpha fusion protein that migrated close to the calculated 94 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.