Routine analysis of proteins by Kjeldahl and Dumas methods: review and interlaboratory study using dairy products

The Kjeldahl and Dumas (combustion) methods were compared in 11 laboratories analyzing samples of milk, skim milk powder, whole milk powder, whey protein concentrate, infant formula, casein, caseinate, 2 reference compounds (glycine and EDTA), and a secondary reference skim milk powder. The comparison was conducted by using international standards where applicable. Overall means were 8.818 g N/100 g by the Kjeldahl method and 8.810 g N/100 g by the Dumas method. No evidence was found for a consistent bias between methods that may be of concern in the trading of dairy produce. A review of more than 10 related trials revealed a lack of consensus in the bias between the 2 methods, suggesting that differences in methodology and sources of systematic error may be contributors. For samples containing > 2 g N/100 g, the Dumas relative repeatability and reproducibility standard deviations were consistently about 0.35 and 0.75%, respectively, whereas the corresponding Kjeldahl values declined generally with N content and were significantly larger. The Dumas precision characteristics may be due to the dominance of Leco analyzers in this trials, and in most other recent trials, rather than an inherent method attribute. Protein determination methods for dairy products need to be reviewed and updated. The Dumas method needs Codex Alimentarius status as a recognized test method.     

Characterization of a negative cis-acting DNA element regulating the transcription of CYP2B1/B2 gene in rat liver

Polymeric films were deposited from hydroxyethylmethacrylate (HEMA) plasma on non-woven poly(butyleneterephtalate) (PBT) filter materials. To test the effect of deposition conditions on surface properties, film were deposited using a constant monomer flow rate and a discharge power ranging from 40-100 W. Surface composition and surface energetics were evaluated by Electron Spectroscopy for Chemical Analysis (ESCA) and contact angle measurement, respectively. Albumin (Alb) and fibrinogen (Fg) adsorption from single protein solutions to the plasma-coated filters was measured. Results illustrate the marked effects of the deposition condition on the surface composition, the surface field of forces, and the protein adsorption behavior. The latter is modeled by the application of the Good-van Oss-Chaudhury theory of Lewis acid-base contribution to interfacial energetics. Materials endowed with widely different properties are obtained from the same monomer and different deposition conditions, a result that must be taken into account both in the production step, to assure constant quality, and in the development of specifically tailored materials.     

Trends in mortality of childhood-onset insulin-dependent diabetes mellitus in Leicestershire: 1940-1991

The relative risk of death by calendar date of diagnosis was investigated in a population-based incident cohort of 845 (463 males:382 females) IDDM diagnosed in Leicestershire before the age of 17 years between 1940 and 1989. The mortality status of 844 (99.9%) patients was determined as of the 31 December 1991, representing 14,346 person-years of risk. Trends in relative risk of death were investigated using Cox proportional hazards modelling for within cohort comparisons and age/sex and calendar time adjusted standardized mortality ratios (SMR) using generalized linear modelling for external comparisons. Median age at diagnosis was 10 years (range 3 months to 16 years); median duration of diabetes 15 years (range 1-51 years). Forty-four patients had died (5.2%; median age at death 31 years, range 11-51 years). A further four patients died at presentation (within 24 h) from ketoacidosis and are excluded from all analyses. Calendar date of diagnosis was found to be an important predictor of mortality. Adjusting for attained age there was evidence of a decline in relative risk of death with calendar date of diagnosis of 3.4% (95% CI, 0.005-6.9%) per annum, equivalent to a 32% fall per decade (95% CI, 5-51%), or 84% (95% CI, 21-97) from 1940 to 1989. The data are consistent with a large fall in mortality between the 1940s and 1950s representing over 50% of the total reduction in mortality between 1940 and 1991. Neither sex nor age at diagnosis were significant predictors of mortality. Over the study period 1940-89 the SMR (male and female combined) fell from 981 (541-1556) to 238 (60-953) relative to the general population. This population-based study shows that the prognosis for Type 1 (insulin-dependent) diabetes mellitus has improved markedly over the period 1940-1991.     

Down-regulation of glial fibrillary acidic protein expression during acute lytic varicella-zoster virus infection of cultured human astrocytes

The effects of the varicella-zoster virus (VZV) OKA vaccine strain in producing morphologic and antigenic changes in dissociated cultures of human fetal brain was investigated. Cultures containing 80% glial acidic fibrillary protein (GFAP), GFAP+ (positive) astrocytes and 20% GFAP- (negative) fibroblastic-like cells were infected with cell-free VZV OKA at a multiplicity of infection of 0.1 plaque-forming units per cell. Cytopathic effects and significant viral antigen labeling with antibodies against VZV gpl and immediate-early (IE) protein 62 were first detected 6 to 7 days postinfection. Several observations indicated that astrocyte GFAP expression was altered and diminished as a result of VZV infection itself, thereby raising doubts about the utility of combining cell markers and viral antigenic labeling in assessing the susceptibility of neural cell types to viral infection. The down-regulation of GFAP expression by VZV appears to be mediated by early rather than late events in the viral replication cycle and may not be the result of virally induced global shut-off of host cell protein synthesis. Similar observations were made using VZV Ellen, a multipassaged, nonvaccine strain. These observations have potential in vivo implications related to histologic analysis of VZV-infected tissues and disease pathogenesis.     

Cooperation between glutathione depletion and protein synthesis inhibition against naturally occurring neuronal death

It is generally agreed that naturally-occurring neuronal death in developing animals is dependent on the synthesis of proteins. Oxidative stress, as when intracellular concentrations of free radicals are raised or when cell constituents such as membrane lipids or protein thiols are oxidized, is also involved in various types of neuronal death. In the present report, we show that the number of naturally dying retinal cells in the chick embryo can be reduced by intraocular injections of cycloheximide, an inhibitor of protein synthesis. L-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthesis, can either enhance or diminish the cell death, depending on the conditions of treatment. Moreover, when the two inhibitors are combined, L-buthionine-[S,R]-sulfoximine potentiates the neuroprotective effects of cycloheximide. Measurements of retinal glutathione concentration and protein synthesis show the specificity of the treatments: buthionine-sulfoximine diminishes glutathione concentrations but not protein synthesis whereas cycloheximide inhibits protein synthesis without decreasing glutathione concentrations. Naturally-occurring neuronal death thus seems to involve the synthesis of proteins, and is also influenced by oxidative phenomena. Our results extend previous data in tectal-lesioned embryos, and suggest that a moderate, non-lethal oxidative stress can enhance the resistance of ganglion cells that might otherwise have died (spontaneously or following axotomy) owing to insufficient retrograde trophic support.     

Anatomical correction of the exstrophy-epispadias complex: analysis of 34 patients

OBJECTIVE: To evaluate the surgical procedures required for anatomical reconstruction of the bladder and penis in the exstrophy-epispadias complex. PATIENTS AND METHODS: All primary exstrophy-epispadias repairs carried out by one surgeon between 1987 and 1997 were reviewed. Bladder closure consisted of full extraperitoneal mobilization, transpositional omphaloplasty, drainage with ureteric and urethral catheters and immobilization with a 'frog-leg' plaster-cast or 'mermaid' dressings. Osteotomies were always performed when bladder closure was attempted after 37 h of age. Before 1990 the osteotomies were posterior vertical iliac (one patient) and subsequently anterior oblique iliac (10 patients). Pre-peritoneal herniotomies, in the absence of a clinical hernia, were included in the primary procedure after 1992. A modified Cantwell technique was used for epispadias repair and this was undertaken at a median of 16 months after bladder closure (range 6-30). RESULTS: Thirty-four patients (27 male) were reviewed; one patient had a chromosomal abnormality, a deletion in the short arm of chromosome 4. The male infants required a median of four procedures (range 2-5) for bladder closure, epispadias reconstruction and herniotomies, while the females needed a median of two (range 2-5). Complete bladder dehiscence, requiring re-closure with osteotomies, occurred in three cases (9%, two male). There were no dehiscences in the primary osteotomy group. Fistulae after epispadias repair occurred in four patients (17%). The bladder capacity increased to > 60 mL in 10 of 15 males by 36 months after epispadias repair. Only two of seven female infants attained a capacity of > 60 mL. Of the 15 infants who did not undergo herniotomy at primary closure, 13 subsequently developed inguinal hernias (one uni- and 11 bilateral) with incarceration occurring in two. Twelve infants underwent herniotomy at primary closure and six developed subsequent hernias (two uni- and four bilateral; P = 0.05) with documented incarceration in two. CONCLUSIONS: Anatomical correction of the exstrophy-epispadias complex remains challenging, but can be achieved with complication rates of < 20% for each stage. Bladder volumes large enough to permit adequate bladder neck reconstruction can be anticipated after epispadias repair in a large proportion of male infants, but remains small in female infants with low outlet resistance. Inguinal herniotomy at the time of bladder closure significantly reduces the incidence of subsequent herniation, which nevertheless remains high.     

Effect of bile/pancreatic secretions on absorption of radioactive or stable zinc. In vivo and in vitro studies

Biliary/pancreatic (B/P) secretions are a major component of endogenous secretions, and endogenously secreted Zn is a primary means of Zn homeostasis. This study examined whether B/P fluid alters the absorption/reabsorption of Zn and, in doing so, whether this contributes to homeostatic control of Zn. Animal experiments utilized rats fed 10 or 300 micrograms Zn/kg diet. An open-ended gut perfusion study in which 65Zn-labeled B/P fluid or 67Zn-labeled and digested diet found significantly decreased Zn absorption from B/P fluid. Although Zn absorption from both sources was less in animals fed diets higher in Zn, there was no interaction of treatment and diet. Further studies utilizing cultured human colon carcinoma cells (CACO-2) as in vitro models of gut enterocytes found that the presence of B/P fluid significantly decreased Zn retention and/or transport and resulted in a redistribution of cellular Zn after 1200 min of incubation. These studies show that a substance in B/P fluid can decrease the absorption of Zn and also suggest that dietary Zn and Zn associated with B/P secretions are absorbed from distinct pools. However, the lack of an interactive effect with diet, and the amount of time required to see differences in CACO-2 cells, suggest that differences in absorption are not a major contributor to Zn homeostasis.     

Agonist action at D2(long) dopamine receptors: ligand binding and functional assays

Clinical pathways are being introduced by hospitals to reduce costs and control unnecessary variation in care. We studied 766 inpatients to measure the impact of a perioperative clinical pathway for patients undergoing knee replacement surgery on hospital costs. One hundred twenty patients underwent knee replacement surgery before the development of a perioperative clinical pathway, and 63 patients underwent knee replacement surgery after pathway implementation. As control groups, we contemporaneously studied 332 patients undergoing radical prostatectomy (no clinical pathway in place for these patients) and 251 patients undergoing hip replacement surgery without a clinical pathway (no clinical pathway and same surgeons as patients having knee replacement surgery). Total hospitalization costs (not charges), excluding professional fees, were computed for all patients. Mean (+/-SD) hospital costs for knee replacement surgery decreased from $21,709 +/- $5985 to $17,618 +/- $3152 after implementation of the clinical pathway. The percent decrease in hospitalization costs was 1.56-fold greater (95% confidence interval 1.02-2.28) in the knee replacement patients than in the radical prostatectomy patients and 2.02-fold greater (95% confidence interval 1.13-5.22) than in the hip replacement patients. If patient outcomes (e.g., patient satisfaction) remain constant with clinical pathways, clinical pathways may be a useful tool for incremental improvements in the cost of perioperative care. Implications: Doctors and nurses can proactively organize and record the elements of hospital care results in a clinical pathway, also known as "care pathways" or "critical pathways." We found that implementing a clinical pathway for patients undergoing knee replacement surgery reduced the hospitalization costs of this surgery.     

Demonstration of the feasibility of emergency department immunization against influenza and pneumococcus

STUDY OBJECTIVE: To demonstrate the feasibility of systematic immunization against influenza and pneumococcus in a public emergency department. METHODS: This was a demonstration project conducted from October 21, 1996, through December 2, 1996, at Cook County Hospital, an inner-city hospital with a 1996 adult ED census of 120,449. Seventy-eight percent of patients are uninsured; 92% are people of color; 73% deny having a primary physician. Only 15% have emergency complaints. Nurses received standing orders that all nonemergency adult patients meeting Centers for Disease Control and Prevention criteria for high risk should be offered immunization against influenza and pneumococcus at triage. Cash prizes were offered to nurses appropriately immunizing the most patients. The date of immunization was entered into the computerized patient registration system, available to all providers within the county system. From November 4 through November 18, an extra nurse was assigned to triage to test for improvement in immunization rates. A time-motion study determined the time required per immunization on the basis of a convenience sample of 8 nurses drawn from all 3 shifts. RESULTS: Only 3% of identified high-risk patients reported previous pneumococcal immunization. Despite extreme variation in nurse performance, 2,631 patients (24% of patients triaged) were screened, and 716 high-risk patients were identified (27% of patients screened). A total of 1234 patients were immunized against influenza, and 241 patients were appropriately immunized against pneumococcus. Sixty-one percent of high-risk patients with no contraindication to influenza immunization were immunized against influenza. Thirty-five percent of high-risk patients not previously immunized against pneumococcus were immunized against pneumococcus. Immunizations per shift per triage nurse varied from 0 to 24. Median time for all activities related to immunization was 4 minutes (range, 2 to 10 minutes). There was no increase in immunization rates with the addition of an extra nurse at triage (95% confidence interval for odds ratio, .929 to 1.153). CONCLUSION: Systematic immunization against influenza and pneumococcus is both needed and feasible in a public ED. "Buy-in" by nurses is variable. Increased staffing alone does not improve immunization rates.     

Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity

Consistent with their role in host defense, mature dendritic cells (DCs) from central lymphoid organs preferentially prime for T helper cell type 1 (Th1)-polarized immunity. However, the "default" T helper response at mucosal surfaces demonstrates Th2 polarity, which is reflected in the cytokine profiles of activated T cells from mucosal lymph nodes. This study on rat respiratory tract DCs (RTDCs) provides an explanation for this paradox. We demonstrate that freshly isolated RTDCs are functionally immature as defined in vitro, being surface major histocompatibility complex (MHC) II lo, endocytosishi, and mixed lymphocyte reactionlo, and these cells produce mRNA encoding interleukin (IL)-10. After ovalbumin (OVA)-pulsing and adoptive transfer, freshly isolated RTDCs preferentially stimulated Th2-dependent OVA-specific immunoglobulin (Ig)G1 responses, and antigen-stimulated splenocytes from recipient animals produced IL-4 in vitro. However, preculture with granulocyte/macrophage colony stimulating factor increased their in vivo IgG priming capacity by 2-3 logs, inducing production of both Th1- and Th2-dependent IgG subclasses and high levels of IFN-gamma by antigen-stimulated splenocytes. Associated phenotypic changes included upregulation of surface MHC II and B7 expression and IL-12 p35 mRNA, and downregulation of endocytosis, MHC II processing- associated genes, and IL-10 mRNA expression. Full expression of IL-12 p40 required additional signals, such as tumor necrosis factor alpha or CD40 ligand. These results suggest that the observed Th2 polarity of the resting mucosal immune system may be an inherent property of the resident DC population, and furthermore that mobilization of Th1 immunity relies absolutely on the provision of appropriate microenvironmental costimuli.