A typical diffusion monitored by flow cytometry: slow diffusion of small molecules in polyelectrolyte multilayers.

An innovative approach has been developed to measure small molecule diffusion in polyelectrolyte multilayers (PEMs) assembled on colloidal particles by means of flow cytometry (FACS). FACS allows changes in fluorescence emission as a function of time to be recorded per particle in a colloidal dispersion. Dithionite, S2O42-, diffusion in PEMs composed of polyallylamine hydrochloride (PAH) and poly styrene sulfonate (PSS) assembled on silica particles has been studied by recording the quenching of (7-nitrobenz-2-oxa-1,3-diazol-4yl)amino (NBD) labelled PAH layers by FACS. NBD is reduced when it encounters dithionite, and is therefore no longer fluorescent. The decay in fluorescence will be used to follow the kinetics of dithionite diffusion. The fluorescence decay curves show slow diffusion that does not follow classical Fickean law. However, by assuming that the diffusion coefficient is time dependent and follows an inverse power law in an atypical diffusion case, it was possible to obtain an excellent fit for the decay curves.

Influence of surface coating on the intracellular behaviour of gold nanoparticles: a fluorescence correlation spectroscopy study.

In the biomedical applications of nanoparticles (NPs), the proper choice of surface chemistry is a crucial aspect in their design. The nature of the coating can heavily impact the interaction of NPs with biomolecules, affect the state of aggregation, and ultimately determine their biological fate. As such, protein corona formation and the aggregation behaviour of gold NPs (Au NPs) are studied here. Au NPs are prepared with four distinct surface functionalisations, namely mercaptosuccinic acid (MSA), N-4-thiobutyroil glucosamine, HS-PEG5000 and HS-alkyl-PEG600. Corona formation, aggregation, and the intracellular behaviour of the Au NPs are then investigated by means of Fluorescence Correlation Spectroscopy (FCS) in cell culture media and in live cells. To evaluate the state of aggregation and the formation of a protein corona, the Au NPs are incubated in cell media and the diffusion coefficient is determined via FCS. The in vitro behaviour is compared with the level of aggregation of the NPs in cells. Diffusion times of the NPs are estimated at different positions in the cell after a one hour incubation period. It is found that the majority of MSA and glucose-Au NPs are present inside the cell as slowly diffusing species with diffusion times (τD) greater than 6000 µs (hydrodynamic diameter >250 nm). PEGylated Au NPs adsorb a small amount of protein and manifest low agglomeration both in media and in living cells. In particular, the HS-alkyl-PEG600 coating shows an excellent correlation between lower protein adsorption, 4-fold lower compared to the MSA coated NPs, and limited intracellular aggregation. In the case of single HS-alkyl-PEG600 coated NPs, it is found that typical intracellular τD values range from 500 to 1500 µs, indicating that these particles display reduced aggregation in the intracellular environment.

Limitations to the use of solid-phase microextraction for quantitation of mixtures of volatile organic sulfur compounds.

A study of the range of volatile organic sulfur compounds produced by brassica plants has highlighted limitations to the use of Carboxen/PDMS fibers for their analysis by solid-phase microextraction (SPME). These fibers are sometimes advocated for the analysis of sulfur gases, but a quantitative comparison of analytical data derived by SPME and by direct gas sampling of standard mixtures of volatile low molecular weight sulfur compounds at 0.01-10 mg/L has identified potential errors associated with their use. Higher molecular compounds displace lower molecular weight compounds as a consequence of competition for active sites on the fiber, and the relative proportions of the components adsorbed onto the fiber depend on their ratio in the headspace. As their relative concentrations change from sample to sample, the varying interactions result in irregular analytical responses, reflected in erratic calibration curves. Standards containing single components are not valid; only a standard containing all components found in the sample to be analyzed, and at the same relative concentrations, is appropriate. In practice, this may preclude the use of the fibers for quantitative analysis of multicomponent mixtures.

Invasion genes are not required for Salmonella enterica serovar typhimurium to breach the intestinal epithelium: evidence that salmonella pathogenicity island 1 has alternative functions during infection.

Salmonella enterica serovar Typhimurium invasion genes are necessary for bacterial invasion of intestinal epithelial cells and are thought to allow salmonellae to enter and cross the intestinal epithelium during infection. Many invasion genes are encoded on Salmonella pathogenicity island 1 (SPI1), and their expression is activated by HilA, a transcription factor also encoded on SPI1. We have studied the role of Salmonella invasion genes during infection of mice following intragastric inoculation. We have found that strains containing a mutation in hilA or invG were recovered from the intestinal contents, intestinal tissues, and systemic tissues at a lower frequency than their parental wild-type strain. In contrast, a strain in which SPI1 is deleted was recovered from infected mice at a frequency similar to that of its parental wild-type strain. The DeltaSPI1 phenotype indicates that S. enterica does not require invasion genes to cross the intestinal epithelium and infect systemic tissues. This result has forced us to reconsider the long-held belief that invasion genes directly mediate bacterial infection of the intestinal mucosa and traversion of the intestinal barrier during infection. Instead, our results suggest that hilA is required for bacterial colonization of the host intestine. The seemingly contradictory phenotype of the DeltaSPI1 mutant suggests that deletion of another gene(s) encoded on SPI1 suppresses the hilA mutant defect. We propose a model for S. enterica pathogenesis in which hilA and invasion genes are required for salmonellae to overcome a host clearance response elicited by another SPI1 gene product(s).

Pituitary hypoplasia in patients with a mutation in the growth hormone-releasing hormone receptor gene.

BACKGROUND AND PURPOSE: Several anatomic abnormalities of the pituitary gland have been described as occurring in association with congenital growth hormone deficiency, including hypoplasia of the adenohypophysis, truncation of the pituitary stalk, and ectopia of the neurohypophysis. Their pathogenesis, however, is obscure. Normal pituitary development is dependent on the sequential expression of a series of ontogenetic factors. Growth hormone-releasing hormone (GHRH) is known to stimulate somatotroph proliferation, and a dwarf mouse model with a mutant GHRH receptor, the "little mouse," has a small anterior pituitary due to hypoplasia of the somatotrophs. We recently described the human homolog of the little mouse (dwarfism of Sindh), caused by a homozygous nonsense mutation in the GHRH receptor gene in a Pakistani kindred. We investigated MR imaging characteristics to gain information regarding the potential role of GHRH in human pituitary organogenesis. METHODS: MR images of the head were obtained of four affected male patients (age range, 22-29 years). Maximal anterior pituitary dimensions were determined from sagittal and coronal images, and pituitary volumes were estimated from cubic and ellipsoid formulae. The measurements were compared with normative values matched for age and sex. RESULTS: The adenohypophysis was small in each of the four patients. The maximal height for the anterior pituitary was 3 mm in three patients and 2 mm in one (mean +/- SD, 2.75 +/- 0.5 mm), which is significantly (P < .001) less than the expected height of 5.6 +/- 1.0 mm for men in this age group. Estimates of anterior pituitary volume in the patients ranged from 75 to 124 mm3 (104 +/- 21 mm3), which corresponds to 35% to 52% of the normal mean volume corrected for small head size (P < .005). No other cranial abnormalities were identified. CONCLUSION: We describe significant hypoplasia of the adenohypophysis occurring in four dwarfs with a nonsense mutation in the GHRH receptor. In addition to isolated growth hormone deficiency and severe dwarfism, affected patients have anterior pituitary hypoplasia, presumably due to somatotroph maldevelopment. Resistance to GHRH explains the hypoplasia of the adenohypophysis--a feature that contributes to growth hormone deficiency in this syndrome. This is one of the few instances in which the molecular basis of pituitary dysmorphogenesis has been identified.

Anticoagulation and minor oral surgery: should the anticoagulation regimen be altered?

PURPOSE: This study was performed to assess the extent of bleeding in anticoagulated patients undergoing minor oral surgery procedures when compared with patients who stop their anticoagulation regimen before surgery and patients who have never been anticoagulated. MATERIALS AND METHODS: Patients on anticoagulant medications undergoing dentoalveolar surgery procedures either stopped their anticoagulation regimen 72 to 96 hours before the planned surgical intervention or continued their regular regimen throughout the time of surgery. Blood loss was measured by weighing sponges used in the procedures, and groups were compared for differences in blood loss. They were also compared with an additional control group that had never been on anticoagulant therapy. RESULTS: There was no difference in blood loss detected among any of the experimental or control groups. No bleeding complications occurred in any anticoagulated patient. CONCLUSIONS: The data suggest that many patients can safely undergo routine outpatient oral surgical procedures without alteration of their regular therapeutic anticoagulation regimens and without additional medical intervention. However, a larger experimental population may be needed to elucidate the appropriateness of this approach to perioperative care.

Acute Sin Nombre hantavirus infection without pulmonary syndrome, United States.

Hantavirus pulmonary syndrome (HPS) occurs in most infections with Sin Nombre virus and other North American hantaviruses. We report five cases of acute hantavirus infection that did not fit the HPS case definition. The patients had characteristic prodromal symptoms without severe pulmonary involvement. These cases suggest that surveillance for HPS may need to be expanded.

Morbid risk of schizophrenia in first-degree relatives of white and African-Caribbean patients with psychosis

The high rate of schizophrenia among the second-generation African-Caribbean population in Britain has prompted much concern and speculation. Sugarman and Craufurd have reported that the morbid risk in the siblings of second-generation African-Caribbean schizophrenic patients was unusually high compared with that of the siblings of white patients. METHOD: We sought to replicate these findings by comparing the morbid risk for schizophrenia in the first-degree relatives of 111 white and 73 African-Caribbean psychotic probands. The latter comprised 35 first-generation (born in the Caribbean) and 38 second-generation (born in Britain) probands. RESULTS: The morbid risk for schizophrenia was similar for the parents and siblings of white and first-generation African-Caribbean patients, and for the parents of the second-generation African-Caribbean probands. However, the siblings of second-generation African-Caribbean psychotic probands had a morbid risk for schizophrenia that was seven times that of their white counterparts (P = 0.007); similarly, the siblings of second-generation African-Caribbean schizophrenic probands had a morbid risk for schizophrenia that was four times that of their white counterparts (P = 0.05). CONCLUSIONS: These findings replicate those of the earlier report of Sugarman and Craufurd, and suggest either that the second-generation African-Caribbean population in Britain is particularly vulnerable to some environmental risk factors for schizophrenia, or that some environmental factors act selectively on this population in Britain.(AU)

Rare pulmonary manifestation of Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome (EDS) is an inherited disorder of connective tissue with multiple thoracic manifestations. We present an unusual thoracic manifestation of EDS consisting of parenchymal cysts and fibrous and fibroosseous nodules. These manifestations may be related to an abnormal attempt at repair of parenchymal or vascular tears.

Effect of stimulus force on perioral direction discrimination: clinical implications.

Clinicians have used the same instrument (viz, Semmes-Weinstein pressure aesthesiometers [Research Design, Inc, Houston, TX] or "von Frey hairs") for tests of both contact detection and direction discrimination. However, patients' ability to discriminate direction may be underestimated by barely detectable moving stimuli. To determine whether the aesthesiometers underestimate direction discrimination, we evaluated the capacity of 13 normal subjects to distinguish opposing directions provided by 10 different hairs. The hairs were selected to deliver forces below and above the contact-detection threshold. Each was stroked over 1.0 cm of perioral skin at the velocity at which the subject was predicted to best discriminate direction of motion. It was found that valid estimates of perioral direction discrimination can be obtained with appropriately selected aesthesiometers. Specifically, the least stiff hair whose handle displays a manufacturer's marking two units greater than that of the contact-detection "threshold hair" should be used to deliver the moving stimuli. The resultant force applied by this hair will exceed 10 times the subject's contact-detection threshold force. If a less-stiff hair is used, the capacity to distinguish direction may be underestimated.

Lyme disease in northwestern coastal California.

To determine the incidence of physician-diagnosed Lyme disease in an endemic area of California, an active surveillance program was implemented in Lake, Mendocino, Sonoma, and southern Humboldt counties. More than 200 medical care providers were called monthly for their list of suspected cases of Lyme disease. Pertinent information was abstracted from the medical record of each patient. Of 153 cases of possible early Lyme disease ascertained from July 1991 to December 1992, 37% consisted of physician-diagnosed erythema migrans. Only 58% of erythema migrans rashes were at least 5 cm in diameter. An additional 43 patients had suspicious rashes not classified as erythema migrans. Of 166 patients with possible late-stage Lyme disease, 31% had specific clinical symptoms and 75% had a positive serologic test. With an incident case defined as physician-diagnosed erythema migrans of at least 5 cm in diameter, the annual incidence of Lyme disease in northwestern coastal California according to active surveillance only was 5.5 per 100,000. The rate of Lyme disease in California is substantially lower than that in the Atlantic northeastern United States. Many suspected cases of Lyme disease in this endemic area do not meet surveillance criteria, which are intentionally restrictive. Although some of the illnesses not meeting surveillance criteria may be due to infection with Borrelia burgdorferi, it appears that Lyme disease is being overdiagnosed in this area.

Properties of osmolyte fluxes activated during regulatory volume decrease in cultured cerebellar granule neurons.

Efflux pathways for amino acids, K, and Cl activated during regulatory volume decrease (RVD) were characterized in cultured cerebellar granule neurons exposed to hyposmotic conditions. Results of this study favor diffusion pores (presumably channels) over energy-dependent transporters as the mechanisms responsible for the efflux of these osmolytes. The selectivity of osmolyte pathways activated by RVD was assessed by increasing the extracellular concentrations of cations, anions, and amino acids to such an extent that upon opening of the pathway, a permeable compound will enter the cell and block RVD by reducing the efflux of water carried by the exit of intracellular osmolytes. The cationic pathway was found selective for K (and Rb), whereas the anionic pathway was rather unselective being permeable to Cl, nitrate, iodine, benzoate, thiocyanate, and sulfate but impermeable to gluconate. Glutamate and aspartate as K but not as Na salts were permeable through the anion channel. RVD was slightly inhibited by quinidine but otherwise was insensitive to known K channel blockers. RVD was inhibited by 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS), niflumic acid, and dipyridamole. Gramicidin did not affect cell volume in isosmotic conditions but greatly accelerated RVD, suggesting that cell permeability to Cl is low in isosmotic conditions but increases markedly during RVD making K permeability the rate limit of the process. The permeability pathway for amino acids activated during RVD as permeable to short chain alpha- and beta-amino acids, but excluded glutamine and basic amino acids.

Regulatory volume decrease in cultured astrocytes. I. Potassium- and chloride-activated permeability.

Regulatory volume decrease (RVD) in detached cerebellar astrocytes in culture after acute exposure to hyposmolarity was characterized in this and the accompanying paper [H. Pasantes-Morales, R. A. Murray, R. Sanches-Olea, and J. Moran. Am. J. Physiol. 266 (Cell Physiol. 35): C172-C178, 1994]. RVD was independent of extracellular calcium, was accelerated at pH 8-9 and retarded at pH 6, and was reduced at temperatures < 18 degrees C. The cationic pathway activated by hyposmolarity was specific for K+ and Rb+, since RVD was abolished and secondary swelling occurred when these ions replaced Na+. However, Li+, choline, tris(hydroxymethyl)aminomethane, and glucosamine, all as Cl- salts, did not affect RVD. The anion pathway was unselective, since RVD was inhibited when NaCl was replaced by anion K+ salts with a permeability rank of SCN- = I- > NO3- > Cl- > benzoate > acetate >> SO3- > gluconate. RVD was unaffected by bumetanide (50 microM) and weakly inhibited by furosemide (2 mM). Quinidine but not other K+ channel blockers inhibited RVD, and its effect was reversed by gramicidin. RVD was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dipyridamole but not by diphenylamine-2-carboxylate or anthracene-9-carboxylate. These results suggest that diffusion possibly via channels rather than cotransporters is involved in the swelling-activated K+ and Cl- fluxes. Gramicidin did not change astrocyte volume in isosmotic conditions, but greatly accelerated RVD, suggesting that low Cl- permeability in isosmotic conditions markedly increases by swelling, thus making K+ permeability the rate-limiting step for RVD.

Regulatory volume decrease in cultured astrocytes. II. Permeability pathway to amino acids and polyols.

The permeability of the hyposmolarity-activated pathway to amino acids and polyols in cultured astrocytes was examined following the change in rate and direction of regulatory volume decrease (RVD) when the extracellular concentration of the osmolytes was increased to reverse their intracellular-extracellular concentration gradient. Activation of the pathway by swelling would allow those permeable osmolytes to enter the cell and inhibit RVD. The pathway was found to be permeable to neutral amino acids, with beta-amino acids (beta-alanine = taurine > gamma-aminobutyric acid) more permeable than alpha-amino acids. Glycine, alanine, threonine, phenylalanine, and asparagine, but not glutamine, were permeable through this pathway. Aspartate was more permeable than glutamate, and K+ and not Na+ must be the accompanying cation. Basic amino acids were excluded. The dimension of the amino acid pore activated by hyposmolarity seems to be at the limit of glutamate-glutamine size. Influx rather than efflux of amino acids was observed when extracellular concentration was greater than intracellular concentration, with differences in the amount accumulated by cells correlating with their efficiency as RVD blockers. Influx of taurine (as representative of permeable amino acids) was inhibited by the Cl- channel blockers/exchangers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (40%) and dipyridamole (85%) , and it is suggested that amino acids permeate through an anion channel. Sorbitol and mannitol, but not inositol, exhibited a small inhibitory effect on the later phase of RVD, whereas inositol slightly accelerated RVD.

A suburban focus of endemic typhus in Los Angeles County: association with seropositive domestic cats and opossums.

Thirty-three cases of locally acquired murine typhus were reported in Los Angeles County residents from May 1984 through February 1988. Only eight cases were reported over the previous 20-year period. Thirty (91%) cases resided within a suburban area encompassing approximately 50 km2 in northcentral Los Angeles or had contact with an animal from this area. Serologic testing (complement fixation and indirect fluorescent antibody) of selected animals in close association with human cases revealed a high prevalence of seropositivity among domestic cats and opossums. Nine (90%) of 10 resident cats tested had demonstrable antibody titers compared with none (0%) of 20 cats from a control area (P < 0.001). Suburban typhus cases were more likely than neighborhood controls to own a cat or dog (odds ratio = 6.9, 95% confidence interval = 1.8, 25.9, P = 0.002). Sixteen (42%) of 38 opossums trapped in close proximity to the residences of cases were seropositive versus none (0%) of 36 opossums from control areas (P < 0.001) A low frequency (2.8%) of seropositivity was found in commensal rodents, and the classic vector of murine typhus, Xenopsylla cheopis, was not found. Ectoparasite indices form seropositive opossums revealed heavy infestations with the cat flea, Ctenocephalides felis (mean flea count = 104.7), a species that readily bites humans. These data provide evidence that a suburban focus of murine typhus exists in Los Angeles that differs substantially from the classic transmission cycle, and that cats, opossums and C. felis may play an important role in the occurrence of human cases.

Dodge/Sweet's construction volume outlook for 1993.

Recovery of the construction industry from its post-'80s crash was never going to be "business as usual." The excess of the previous decade--commercial overbuilding, the banking crisis, the federal-budget deficit--all meant that the upturn would take place against a backdrop unlike any seen before. Indeed, cyclical recovery within the context of structural change is a good way to look at what has happened so far in 1992, and what is expected to occur over the next several years.

Assessment of epithelial thickness by ultrasonic imaging.

Moisturizing creams and lotions are an important aspect of preventing pressure ulcers. This study reports the increased thickening of the epithelium on 11 subjects who used a moisturizing cream (treatment) for 20 days. The thickening of the epithelium was measured before use of the moisturizing cream, on day 1, day 8, and day 20 of the treatment, and 12 days after the treatment. The ultrasound measurements were similar before the treatment and 12 days post treatment; the measurements increased for the days of treatment. Current research would lead us to believe that short-term increase in epithelial thickness as produced by a moisturizer is a result of increased water content. Ultrasound imaging may be the new standard for comparing the effects of moisturing products.

Mycobacterium chelonae infection among patients receiving high-flux dialysis in a hemodialysis clinic in California.

Between July 1987 and January 1988, five patients dialyzed at a hemodialysis outpatient clinic developed systemic Mycobacterium chelonae abscessus (MCA) infections. Four of the five patients had arteriovenous graft infections, and two died during antimicrobial therapy. Case-patients were more likely than control-patients to have received high-flux dialysis during the 6 mo before their infection (100% vs. 30%, P = .009). MCA was cultured from the hose connected to a water spray device used for manual reprocessing of high-flux but not regular dialyzers. Renalin was the germicide used to manually disinfect dialyzers for reuse and was prepared at a concentration of 2.5%. Laboratory studies documented survival of MCA from two high-flux dialyzers that were reprocessed in a manner similar to that used in the clinic. Early diagnosis with complete rather than partial graft removal in combination with antimicrobial therapy is recommended for MCA graft infections. In addition, 2.5% Renalin did not appear to ensure complete killing of MCA in high-flux dialyzers that were manually reprocessed at this hemodialysis clinic.

Description and validation of an in situ autoperfusion method to determine nutrient absorption and metabolism in bovine small intestine.

Holstein bull calves, 8 to 12 wk of age, were anesthetized with halothane gas. An approximate 20-cm section of small intestine, 60 to 90 cm proximal to the ileocecal junction was clamped to isolate blood circulation to a single set of arcuate vessels and to form an intestinal segment fitted for infusion and drainage. The vein was catheterized to allow total venous collection. Donor blood was transfused via jugular vein to replace venous drainage. This technique was evaluated in four calves by exposing the lumen to eight replications (12 or 20 min incubation, 30-min wash with 39 C saline) of 16 mM L-Met (14C-labeled). Time course appearance of Met in venous blood indicated similar rates and patterns of absorption for individual calves. There were no clinically significant alterations in jugular blood chemistry profiles across replications. Four calves were used to evaluate the effect of three isotonic perfusion media (saline, Krebs-Ringer bicarbonate and M-199 tissue culture media) on Lys and Met absorption. Venous flow rates and absorption of Lys were faster with Krebs buffer than with other media. Perfusate medium did not influence venous flow rates or absorption of Met. Effect of restricting venous flow on absorption of Lys and Met was evaluated in two calves. Flow was alternately controlled (6.5 ml/min) or allowed to flow freely (mean = 12.2 ml/min). Restricting flow decreased steady-state absorption. Light and scanning microscopy indicated maintenance of mucosal tissue integrity throughout 8 h of anesthesia. Results demonstrate validity of the in situ technique to study nutrient absorption in the young bovine.

A micro-technique for preparing homogenates of biological samples.

A method is described for preparing homogenates of insects, mites, nematodes, or fungal spores for electrophoretic analysis which overcomes the disadvantages of existing methods. It is also applicable to the homogenization of microsamples of animal tissue. The method involves grinding the samples in glass tubes, closed at one end, prepared from melting-point capillaries. A range of plungers is fabricated from the stainless-steel plungers from broken microsyringes, or from stainless-steel wire. The plunger is operated by hand to homogenize a sample in 2-20 microliter of buffer in a tube. The techniques for handling the samples before and after homogenization and for labeling and centrifuging them are described. Compared with existing methods, the procedure minimizes sample loss and risk of cross-contamination and eliminates the possibility of overheating the sample during homogenization.