Influence of multi-lumen extensions on fluid flow through intravenous cannulae.

Multi-lumen extensions used to infuse multiple fluids via a single intravenous cannula might increase resistance and so limit the flow that can be achieved. We constructed low-pressure and high-pressure models and compared the effect of two different multi-lumen extensions on flow rate. Both multi-lumen extensions reduced flows by up to 76% (p < 0.001). The effect was greatest with large cannulae and in the high-pressure model, with the longer and narrower extension most impeding flow. Multi-lumen extensions can therefore significantly impede fluid flow, and should be avoided or removed when rapid infusion is required. These effects are less important in paediatric anaesthesia where smaller cannulae are used. Manufacturers should include internal diameter or flow effects on the packaging of these extensions to assist clinicians in making such decisions.

Expression of muscarinic receptor subtypes in tree shrew ocular tissues and their regulation during the development of myopia.

PURPOSE: Muscarinic receptors are known to regulate several important physiologic processes in the eye. Antagonists to these receptors such as atropine and pirenzepine are effective at stopping the excessive ocular growth that results in myopia. However, their site of action is unknown. This study details ocular muscarinic subtype expression within a well documented model of eye growth and investigates their expression during early stages of myopia induction. METHODS: Total RNA was isolated from tree shrew corneal, iris/ciliary body, retinal, choroidal, and scleral tissue samples and was reverse transcribed. Using tree shrew-specific primers to the five muscarinic acetylcholine receptor subtypes (CHRM1-CHRM5), products were amplified using polymerase chain reaction (PCR) and their identity confirmed using automated sequencing. The expression of the receptor proteins (M1-M5) were also explored in the retina, choroid, and sclera using immunohistochemistry. Myopia was induced in the tree shrew for one or five days using monocular deprivation of pattern vision, and the expression of the receptor subtypes was assessed in the retina, choroid, and sclera using real-time PCR. RESULTS: All five muscarinic receptor subtypes were expressed in the iris/ciliary body, retina, choroid, and sclera while gene products corresponding to CHRM1, CHRM3, CHRM4, and CHRM5 were present in the corneal samples. The gene expression data were confirmed by immunohistochemistry with the M1-M5 proteins detected in the retina, choroid, and sclera. After one or five days of myopia development, muscarinic receptor gene expression remained unaltered in the retinal, choroidal, and scleral tissue samples. CONCLUSIONS: This study provides a comprehensive profile of muscarinic receptor gene and protein expression in tree shrew ocular tissues with all receptor subtypes found in tissues implicated in the control of eye growth. Despite the efficacy of muscarinic antagonists at inhibiting myopia development, the genes of the muscarinic receptor subtypes are neither regulated early in myopia (before measurable axial elongation) nor after significant structural change.

A functional network module for Smith-Magenis syndrome.

Disorders with overlapping diagnostic features are grouped into a network module. Based on phenotypic similarities or differential diagnoses, it is possible to identify functional pathways leading to individual features. We generated a Smith-Magenis syndrome (SMS)-specific network module utilizing patient clinical data, text mining from the Online Mendelian Inheritance in Man database, and in vitro functional analysis. We tested our module by functional studies based on a hypothesis that RAI1 acts through phenotype-specific pathways involving several downstream genes, which are altered due to RAI1 haploinsufficiency. A preliminary genome-wide gene expression study was performed using microarrays on RAI1 haploinsufficient cells created by RNAi-based approximately 50% knockdown of RAI1 in HEK293T cells. The top dysregulated genes were involved in growth signaling and insulin sensitivity, neuronal differentiation, lipid biosynthesis and fat mobilization, circadian activity, behavior, renal, cardiovascular and skeletal development, gene expression, and cell-cycle regulation and recombination, reflecting the spectrum of clinical features observed in SMS. Validation using real-time quantitative reverse transcriptase polymerase chain reaction confirmed the gene expression profile of 75% of the selected genes analyzed in both HEK293T RAI1 knockdown cells and SMS lymphoblastoid cell lines. Overall, these data support a method for identifying genes and pathways responsible for individual clinical features in a complex disorder such as SMS.

Human erythrocyte shape regulation: interaction of metabolic and redox status.

The echinocyte-to-discocyte shape recovery of metabolically depleted erythrocytes is compromised by sulfhydryl reducing agents (Truong, H.-T.N., Ferrell, J.E., Jr. and Huestis, W.H. (1986) Blood 67, 214-221). In the presence of dithiothreitol (DTT) and sugars, crenated cells recover normal discoid shape transiently, but then develop the invaginations and intracellular inclusions of stomatocytes. The stomatogenic effects of DTT were investigated in erythrocytes recovering from crenation induced by several independent mechanisms. Cells crenated by direct manipulation of the membrane bilayer (lysophosphatidylcholine incorporation) recovered discoid shape similarly in the presence and absence of the reducing agent. In contrast, resealed ghosts and cells crenated by Mg2+ depletion or Ca2+ loading did not maintain stable discoid morphology in the presence of DTT, proceeding further to form stomatocytes. Thus cell crenation by expedients that involve cellular metabolic processes develop a redox-related morphological instability that is not found in amphipath-crenated cells.

Dithiothreitol stimulates the activity of the plasma membrane aminophospholipid translocator.

Metabolic depletion induces human erythrocytes to crenate, a shape change that is reversed when ATP is regenerated by nutrient supplementation. In the presence of the sulfhydryl reducing agent dithiothreitol (DTT), this shape reversal is exaggerated, proceeding beyond normal discoid morphology to stomatocytic forms. DTT-induced stomatocytosis does not correlate consistently with alterations in cell ATP, spectrin phosphorylation, or phosphoinositide metabolism (Truong, H.-T.N., Ferrell, J.E., Jr. and Huestis, W.H. (1986) Blood 67, 214-221). The effect of DTT on outer-to-inner-monolayer transport of aminophospholipids was examined by monitoring shape changes induced by dilauroylphosphatidylserine (DLPS). Stomatocytosis induced by transport of this exogenous lipid to the membrane inner monolayer is accelerated and exaggerated by DTT. The effect of DTT on DLPS translocation is reversible and temperature dependent, consistent with the intervention of reducing agents in the activity of the aminophospholipid translocator. These findings bear on the relationship between cell redox status and shape regulation.

Anaphylaxis-induced hyperfibrinolysis in pregnancy.

Anaphylaxis during pregnancy is rare but life threatening to both mother and fetus. The anaesthetist may be unexpectedly faced with an obstructing airway, severe bronchospasm and cardiac arrest requiring perimortem caesarean delivery to relieve aortocaval compression. We present a case of anaphylaxis-induced hyperfibrinolysis, an infrequently discussed complication that could exacerbate postpartum haemorrhage and hamper resuscitative efforts.

A 19F-NMR study of the membrane-binding region of D-lactate dehydrogenase of Escherichia coli.

D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65,000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of D-LDH and its interaction with phospholipids. We incorporated 5-fluorotryptophan (5F-Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide-directed mutagenesis, and studied the 5F-Trp-labeled enzymes using 19F-NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin-labeled fatty acid, which broadens the resonance from any residue within 15 A, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F-NMR spectroscopy of wild-type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid. These experiments indicate that 9-10 Phe and 3-4 Tyr residues are located near the lipid phase.

Inhibition of myopia development in chicks using himbacine: a role for M(4) receptors?

The success of the M(1)-selective muscarinic antagonist pirenzepine in preventing myopia development in animal models implicates a role for the M(1) receptor. However, the relatively high dose of pirenzepine required may indicate that the drug acts through another receptor subtype. This study examined whether the M(4)-selective antagonist, himbacine, could also prevent myopia. Daily intravitreal injections of himbacine inhibited the inducement of myopia in chick eyes in a dose- dependent manner. Doses < or = 200 microg caused no significant inhibition of induced myopia compared to controls (-13.7 +/- 2.3 vs -16.2 +/- 0.9D, ANOVA p = 0.37), whilst a dose of 800 microg almost completely inhibited the induced myopia (-2.4 +/- 2.0, p < 0.01). Findings demonstrate himbacine is effective at preventing the development of myopia in chick and implicates a role for the M4 receptor.

Glycosaminoglycan synthesis in the separate layers of the chick sclera during myopic eye growth: comparison with mammals.

PURPOSE: Studies in animal models of refractive development have shown that the development of and recovery from induced myopia is associated with visually-guided changes in scleral glycosaminoglycan synthesis. The present study sought to determine whether differential patterns of scleral glycosaminoglycan synthesis are present in the fibrous scleral layer of the chick during myopia development or recovery, as has previously been reported in the mammalian sclera. METHODS: Myopia was induced in young chicks by monocular deprivation of pattern vision over 5 days. Other animals underwent monocular deprivation, then had the occluder removed and were allowed 2 days of recovery. A group of age-matched normal animals served as a control. Newly synthesised glycosaminoglycans in the scleral layers were labelled in vivo, using a [(35)S]-labelled precursor delivered intraperitoneally on the final experimental day. Incorporation of this sulphate into glycosaminoglycans of the fibrous and cartilaginous scleral layers was assessed in proteinase K digests by selective precipitation with alcian blue. RESULTS: Glycosaminoglycan synthesis in the fibrous scleral layers of myopic and recovering eyes was not significantly different to contralateral control eyes (+14 +/- 7%, p = 0.09 and -2 +/- 4%, p = 0.64 respectively). In contrast, glycosaminoglycan synthesis was significantly elevated, relative to controls, in the cartilaginous scleral layer of eyes developing myopia (+63 +/- 18%, p < 0.02), whereas in recovering eyes there was found to be a significant decrease in synthesis in the cartilaginous layer (-40 +/- 6%, p < 0.001). CONCLUSIONS: The results of the current study demonstrate that the fibrous scleral layer of the chick does not display the characteristic differential patterns of glycosaminoglycan synthesis that are found in the mammalian sclera during myopia development and recovery. However, as has previously been reported, the cartilaginous layer of the chick sclera does display differential glycosaminoglycan expression, although the direction of regulation is opposite to that found in the fibrous sclera of mammals.

Interaction of the membrane-bound D-lactate dehydrogenase of Escherichia coli with phospholipid vesicles and reconstitution of activity using a spin-labeled fatty acid as an electron acceptor: a magnetic resonance and biochemical study.

The interaction with phospholipid vesicles of the membrane-bound respiratory enzyme D-lactate dehydrogenase of Escherichia coli has been studied. Proteolytic digestion studies show that D-lactate dehydrogenase is protected from trypsin digestion to a larger extent when it interacts with phosphatidylglycerol than with phosphatidylcholine vesicles. Wild-type D-lactate dehydrogenase and mutants in which an additional tryptophan is substituted in selected areas by site-specific oligonucleotide-directed mutagenesis have been labeled with 5-fluorotryptophan. 19F nuclear magnetic resonance studies of the interaction of these labeled enzymes with small unilamellar phospholipid vesicles show that Trp 243, 340, and 361 are exposed to the lipid phase, while Trp 384, 407, and 567 are accessible to the external aqueous phase. Reconstitution of enzymatic activity in phospholipid vesicles has been studied by adding enzyme and substrate to phospholipid vesicles containing a spin-labeled fatty acid as an electron acceptor. The reduction of the doxyl group of the spin-labeled fatty acid has been monitored indirectly by nuclear magnetic resonance and directly by electron paramagnetic resonance. These results indicate that an artificial electron-transfer system can be created by mixing D-lactate dehydrogenase and D-lactate together with phospholipid vesicles containing spin-labeled fatty acids.

Sulfhydryl reducing agents and shape regulation in human erythrocytes.

Metabolic crenation of red cells is reversible; on addition of nutrients, echinocytes recover the normal discoid shape. When the shape recovery takes place in the presence of reducing agents such as dithiothreitol (DTT), morphological change continues until the cells are stomatocytic. The degree of stomatocytosis varies, depending on the cell morphology when the nutrients and reducing agent are added. DTT has minimal effect on the shape of normal discocytes, but in its presence, mildly echinocytic cells become slightly cupped and advanced-stage echinocytes become severely stomatocytic. DTT must be present continuously for development and retention of stomatocytosis; echinocytes preincubated with or metabolically depleted in DTT do not become stomatocytic when supplemented in the absence of DTT, and DTT-induced stomatocytes revert to discocytes when the reducing agent is removed. DTT has no effect on adenosine triphosphate synthesis or equilibrium cell glutathione levels, and the induced stomatocytosis is not inhibited by excluding oxygen from cells during depletion. Spectrin phosphorylation and phosphate turnover are not affected by DTT. The echinocyte-to-discocyte transformation coincides with phosphorylation of membrane inner monolayer lipids (diacylglycerol to phosphatidic acid and phosphatidylinositol to phosphatidylinositol-4,5-bisphosphate). Overphosphorylation of these phospholipids is not responsible for the exaggerated shape recovery seen with reducing agents; phosphorylation of inner monolayer lipids proceeds identically in the presence and absence of DTT.

Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli.

A combination of site-specific mutagenesis and 19F nuclear magnetic resonance has been used to investigate the structural properties of D-lactate dehydrogenase, a membrane-associated enzyme of Escherichia coli. The protein (65,000 Da) has been labeled with 5-fluorotryptophan for 19F nuclear magnetic resonance studies. Tryptophan has been substituted for individual phenylalanine, tyrosine, isoleucine, and leucine residues at various positions throughout the enzyme molecule, and the fluorinated native and substituted tryptophan residues have been used as probes of the local environment. All 24 mutants thus generated are expressed in E. coli. Ten are fully active and purfiable following the usual procedure, while 14 either are inactive or produce low levels of activity. The amount of active enzyme produced from the low-yield mutants is dependent on the temperature at which synthesis is carried out, with more active enzyme produced at 18 degrees C than at 27, 35, or 42 degrees C. Cells grown at 27 degrees C and then incubated at 42 degrees C retain 90-100% of their activity. All of the expressed protein from the inactive mutants is Triton-insoluble, aggregated, and not readily purfiable; the inactive mutant protein appears to be improperly folded. Most of the expressed D-lactate dehydrogenase from the partially active mutants is also Triton-insoluble; a small fraction, however, is soluble in Triton and can be purified to yield active enzyme. All the purified enzymes from these low-yield mutants of D-lactate dehydrogenase have essentially normal VmaxS, and all but two have normal KmS. Once purified, the low-yield mutant enzymes are stable at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Risk factors for HIV seropositivity in a sample of drug users in drug treatment in Ho Chi Minh City, Vietnam.

The article describes drug use behaviors, AIDS knowledge, risks for HIV infection, and HIV seroprevalence in drug users entering rehabilitation in Ho Chi Minh City, Vietnam. A cross-sectional, descriptive survey of all new entrants to a residential drug treatment center was conducted with linked HIV serology between July 1 and July 14, 1995. A total of 105 subjects participated: 101 men and 4 women. HIV serostatus was available for 88 subjects. Forty-seven percent (41 of 88 subjects) were HIV-positive. Median age of the subjects was 38 years. Mean length of injection drug use was 13.2 years (range, 1-27 years). The primary drug of injection was opium (96%), although 59% of subjects also injected "Western" drugs such as sedatives or tranquilizers. Eighty-two percent (86 of 105 subjects) correctly answered at least 7 of 10 AIDS knowledge questions, and only 28% (27 of 97 subjects) reported any needle sharing in the last 5 years. Seropositivity was associated with a history of previous treatment for drug abuse (p = 0.002), longer history of injecting drugs (p = 0.003), use of Western drugs (p = 0.03), and higher educational level (p = 0.05). Multivariate analysis found that the independent predictors of HIV seropositivity were history of previous treatment for drug abuse (p = 0.06) and longer history of injecting drugs (p = 0.05). Despite low levels of self-reported needle sharing and high levels of AIDS knowledge, HIV seroprevalence was high in this sample. The potential for epidemic spread of HIV in Vietnamese drug users is substantial. Risk-reduction programs and intense AIDS education projects targeting the population of drug users are necessary to control the AIDS epidemic in Vietnam.

PIP: A cross-sectional survey of all 105 new entrants to a residential drug treatment center in Ho Chi Minh City, Viet Nam, in a 2-week period in 1995 investigated HIV seroprevalence and its risk factors. The median age of study participants was 38 years and the mean length of injection drug use--primarily of opium--was 13.2 years. 86 drug users (82%) correctly answered at least 7 of 10 questions assessing knowledge of AIDS. Only 27 (28%) of the 97 intravenous drug users reported any needle-sharing in the last 5 years. Of the 88 subjects for whom HIV serostatus results were available, 41 (47%) were HIV-positive. Seropositivity was significantly associated with a history of previous treatment for drug use, longer history of injecting drugs, use of Western drugs, and higher educational level. The multivariate analysis identified 2 independent predictors of HIV seropositivity: history of previous treatment for drug abuse (odds ratio (OR), 1.26; 95% confidence interval (CI), 1.00-1.58) and longer history of injecting drugs (OR, 1.06; 95% CI, 1.00-1.14). These findings indicate the potential for epidemic spread of HIV among drug users in Viet Nam and pinpoint a need for risk-reduction programs in this population.

Site-specific incorporation of 5-fluorotryptophan as a probe of the structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli: a 19F nuclear magnetic resonance study.

The structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli have been investigated by fluorine-19 nuclear magnetic resonance spectroscopy of 5-fluorotryptophan-labeled enzyme in conjunction with oligonucleotide-directed, site-specific mutagenesis. 5-Fluorotryptophan has been substituted for nine phenylalanine, tyrosine, and leucine residues in the enzyme molecule without loss of activity. The 19F signals from these additional tryptophan residues have been used as markers for sensitivity to substrate, exposure to aqueous solvent, and proximity to a lipid-bound spin-label. The nuclear magnetic resonance data show that two mutational sites, at amino acid residues 340 and 361, are near the lipid environment used to stabilize the enzyme. There are a number of amino acid residues on the carboxyl side of this region that are strongly sensitive to the aqueous solvent. The environment of the wild-type tryptophan residue at position 469 changes as a result of two of the substitution mutations, suggesting some amino acid residue-residue interactions. Secondary structure prediction methods indicate a possible binding site for the flavin adenine dinucleotide cofactor in the carboxyl end of the enzyme molecule. These results suggest that the membrane-bound D-lactate dehydrogenase may have the two-domain structure of many cytoplasmic dehydrogenases but with the addition of a membrane-binding domain between the catalytic and cofactor-binding domains. This type of three-domain structure may be of general significance for understanding the structure of membrane-bound proteins which do not traverse the lipid bilayer of membranes.

Effect of turbulent-flow pasteurization on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk.

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72 degrees C in duplicate trials. No strains survived at 72 degrees C for 15 s; and only one strain survived at 69 degrees C. Means of pooled D values (decimal reduction times) at 63 and 66 degrees C were 15.0 +/- 2.8 s (95% confidence interval) and 5.9 +/- 0.7 s (95% confidence interval), respectively. The mean extrapolated D72 degrees C was <2.03 s. This was equivalent to a >7 log10 kill at 72 degrees C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6 degrees C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72 degrees C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2 degrees above 72 degrees C.