A polydisperse linear random coil model for the quaternary structure of pig colonic mucin

The distribution of molecular weights for polymeric colonic mucus glycoprotein or ``mucin' isolated and solubilised in the presence of protease inhibitors from pig colons is shown to be considerably greater than its ``subunit' (thiol reduction product) and papain digested forms using the technique of size-exclusion chromatography coupled to multi-angle laser light scattering, and confirmed by sedimentation equilibrium measurements. The conformation of this mucin is probed by examining the molecular weight – intrinsic viscosity relationship in terms of the Mark-Houwink-Kuhn-Sakurada analysis for its polymeric (or ``whole'), reduced and papain-digested forms: an exponent ``a' of (1.1±0.1) is obtained indicating a linear random coil conformation consistent with other mucins. Size-exclusion chromatography coupled to multi-angle laser light scattering is shown to provide a relatively simple complementary technique to sedimentation equilibrium for the molecular weight distribution analysis of polydisperse materials. Received: 29 November 1996 / Accepted: 2 December 1996     

Effect of carbon:nitrogen ratio on kinetics of phenol biodegradation byAcinetobacter johnsonii in saturated sand

In polluted soil or ground water, inorganic nutrients such as nitrogen may be limiting, so that Monod kinetics for carbon limitation may not describe microbial growth and contaminant biodegradation rates. To test this hypothesis we measured¹⁴CO₂ evolved by a pure culture ofAcinetobacter johnsonii degrading 120 µg¹⁴C-phenol per ml in saturated sand with molar carbon:nitrogen (CN) ratios ranging from 1.5 to 560. We fit kinetics models to the data using non-linear least squares regression. Phenol disappearance and population growth were also measured at CN1.5 and CN560.After a 5- to 10-hour lag period, most of the¹⁴CO₂ evolution curves at all CN ratios displayed a sigmoidal shape, suggesting that the microbial populations grew. As CN ratio increased, the initial rate of¹⁴CO₂ evolution decreased. Cell growth and phenol consumption occurred at both CN1.5 and CN560, and showed the same trends as the¹⁴CO₂ data. A kinetics model assuming population growth limited by a single substrate best fit the¹⁴CO₂ evolution data for CN1.5. At intermediate to high CN ratios, the data were best fit by a model originally formulated to describe no-growth metabolism of one substrate coupled with microbial growth on a second substrate. We suggest that this dual-substrate model describes linear growth on phenol while nitrogen is available and first-order metabolism of phenol without growth after nitrogen is depleted.     

Effect of Sex, Menstrual Cycle Phase, and Oral Contraceptive Use on Circadian Temperature Rhythms

The circadian rhythm of rectal temperature was continuously recorded over several consecutive days in young men and women on regular nocturnal sleep schedules. There were 50 men, 21 women with natural menstrual cycles [i.e., not taking oral contraceptives (OCs) (10 in the follicular phase and 11 in the luteal phase)], and 14 women using OCs (6 in the pseudofollicular phase and 8 in the pseudoluteal phase). Circadian phase and amplitude were estimated using a curve-fitting procedure, and temperature levels were determined from the raw data. A two-way analysis of variance (ANOVA) on the data from the four groups of women, with factors menstrual cycle phase (follicular, luteal) and OC use (yes, no), showed that temperature during sleep was lower during the follicular phase than during the luteal phase. Since waking temperatures were similar in the two phases, the circadian amplitude was also larger during the follicular phase. The lower follicular phase sleep temperature also resulted in a lower 24-h temperature during the follicular phase. The two-way ANOVA showed that temperature during sleep and 24-h temperature were lower in naturally cycling women than in women taking OCs. A one-way ANOVA on the temperature rhythm parameters from the five groups of subjects showed that the temperature rhythms of the men and of the naturally cycling women in the follicular phase were not significantly different. Both of these groups had lower temperatures during sleep, lower 24-h temperatures, and larger circadian amplitudes than the other groups. There were no significant differences in circadian phase among the five groups studied. In conclusion, menstrual cycle phase, OC use, and sex affect the amplitude and level, but not the phase, of the overt circadian temperature rhythm.     

A standard tissue as a control for histochemical and immunohistochemical staining

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel^? and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.     

Effects of bottle size in determinations of primary productivity by phytoplankton

Determinations of primary productivity were made by the radiocarbon method at regular intervals over a year in samples, taken from the eutrophic waters of the Menai Strait, North Wales, and contained in bottles of capacities from 135 ml up to 2290 ml. Fixation per unit volume in particulate matter retained by 0 · 45 m pore size membrane filters was independent of bottle size over most of the year. There was a consistent divergence, however, during the period of rapid phytoplankton growth in the spring, when small bottles gave up to three times higher values than large ones. Possible effects of bottle size on primary productivity determinations are discussed but the cause of this particular result is not apparent.     

Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.     

Preflight adjustment to eastward travel: 3 days of advancing sleep with and without morning bright light

Jet lag is caused by a misalignment between circadian rhythms and local destination time. As humans typically take longer to re-entrain after a phase advance than a phase delay, eastward travel is often more difficult than westward travel. Previous strategies to reduce jet lag have focused on shaping the perceived light-dark cycle after arrival, in order to facilitate a phase shift in the appropriate direction. Here we tested treatments that travelers could use to phase advance their circadian rhythms prior to eastward flight. Thus, travelers would arrive with their circadian rhythms already partially re-entrained to local time. We determined how far the circadian rhythms phase advanced, and the associated side effects related to sleep and mood. Twenty-eight healthy young subjects participated in 1 of 3 different treatments, which all phase advanced each subject's habitual sleep schedule by 1 h/day for 3 days. The 3 treatments differed in morning light exposure for the 1st 3.5 h after waking on each of the 3 days: continuous bright light (> 3000 lux), intermittent bright light (> 3000 lux, 0.5 h on, 0.5 off, etc.), or ordinary dim indoor light (< 60 lux). A phase assessment in dim light (< 10 lux) was conducted before and after the treatments to determine the endogenous salivary dim light melatonin onset (DLMO). The mean DLMO phase advances in the dim, intermittent, and continuous light groups were 0.6, 1.5, and 2.1 h, respectively. The intermittent and continuous light groups advanced significantly more than the dim light group (p < 0.01) but were not significantly different from each other. The side effects as assessed with actigraphy and logs were small. A 2-h phase advance may seem small compared to a 6- to 9-h time zone change, as occurs with eastward travel from the USA to Europe. However, a small phase advance will not only reduce the degree of re-entrainment required after arrival, but may also increase postflight exposure to phase-advancing light relative to phase-delaying light, thereby reducing the risk of antidromic re-entrainment. More days of preflight treatment could be used to produce even larger phase advances and potentially eliminate jet lag.     

Combinations of bright light, scheduled dark, sunglasses, and melatonin to facilitate circadian entrainment to night shift work

Various combinations of interventions were used to phase-delay circadian rhythms to correct their misalignment with night work and day sleep. Young participants (median age = 22, n = 67) participated in 5 consecutive simulated night shifts (2300 to 0700) and then slept at home (0830 to 1530) in darkened bedrooms. Participants wore sunglasses with normal or dark lenses (transmission 15% or 2%) when outside during the day. Participants took placebo or melatonin (1.8 mg sustained release) before daytime sleep. During the night shifts, participants were exposed to a moving (delaying) pattern of intermittent bright light (approximately 5000 lux, 20 min on, 40 min off, 4-5 light pulses/night) or remained in dim light (approximately 150 lux). There were 6 intervention groups ranging from the least complex (normal sunglasses) to the most complex (dark sunglasses + bright light + melatonin). The dim light melatonin onset (DLMO) was assessed before and after the night shifts (baseline and final), and 7 h was added to estimate the temperature minimum (Tmin). Participants were categorized by their amount of reentrainment based on their final Tmin: not re-entrained (Tmin before the daytime dark/sleep period), partially re-entrained (Tmin during the first half of dark/sleep), or completely re-entrained (Tmin during the second half of dark/ sleep). The sample was split into earlier participants (baseline Tmin < or = 0700, sunlight during the commute home fell after the Tmin) and later participants (baseline Tmin > 0700). The later participants were completely re-entrained regardless of intervention group, whereas the degree of re-entrainment for the earlier participants depended on the interventions. With bright light during the night shift, almost all of the earlier participants achieved complete re-entrainment, and the phase delay shift was so large that darker sunglasses and melatonin could not increase its magnitude. With only room light during the night shift, darker sunglasses helped earlier participants phase-delay more than normal sunglasses, but melatonin did not increase the phase delay. The authors recommend the combination of intermittent bright light during the night shift, sunglasses (as dark as possible) during the commute home, and a regular, early daytime dark/sleep period if the goal is complete circadian adaptation to night-shift work.     

The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase

The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation.