Complete Genome Sequences of Nine Mycobacteriophages from New Zealand, Beatrix, Carthage, Daegal, Dulcie, Fancypants, Fenn, Inca, Naira, and Robyn.

Beatrix, Carthage, Daegal, Dulcie, Fancypants, Fenn, Inca, Naira, and Robyn are newly isolated bacteriophages capable of infecting Mycolicibacterium smegmatis mc2 155. We discovered, sequenced, and annotated these New Zealand bacteriophages. These phages illustrate that New Zealand harbors a selection of the highly diverse and distributed mycobacteriophage clusters found globally.

The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii.

The ability of intracellular parasites to monitor the viability of their host cells is essential for their survival. The protozoan parasite Toxoplasma gondii actively invades nucleated animal cells and replicates in their cytoplasm. Two to 3 days after infection, the parasite-filled host cell breaks down and the parasites leave to initiate infection of a new cell. Parasite egress from the host cell is triggered by rupture of the host plasma membrane and the ensuing reduction in the concentration of cytoplasmic potassium. The many other changes in host cell composition do not appear be used as triggers. The reduction in the host cell [K(+)] appears to activate a phospholipase C activity in Toxoplasma that, in turn, causes an increase in cytoplasmic [Ca(2+)] in the parasite. The latter appears to be necessary and sufficient for inducing egress, as buffering of cytoplasmic Ca(2+) blocks egress and calcium ionophores circumvent the need for a reduction of host cell [K(+)] and parasite phospholipase C activation. The increase in [Ca(2+)](C) brings about egress by the activation of at least two signaling pathways: the protein kinase TgCDPK1 and the calmodulin-dependent protein phosphatase calcineurin.

Reduced expression of connexin-43 and functional gap junction coupling in human gliomas.

Gap junctions are an important means for intercellular communication during development, processes of tissue differentiation, and in maintenance of adult tissue homeostasis. We investigated the expression levels and distribution of connexin-43 (Cx-43), the most abundant astrocytic gap junction protein, in acutely isolated astrocytes and glioma cells from biopsy tissue obtained from patients diagnosed with glioblastoma multiforme (GBM), low-grade astrocytomas (LGAs), or mesial temporal lobe epilepsy. Western blot and immunohistochemical analyses indicated an inverse correlation between the amount of Cx-43 protein and tumor malignancy grade, as assessed by calculating tissue mitotic indexes (MI) obtained using anti-Ki-67 nuclear antigen staining. Samples from epilepsy patients had a low MI and were intensely positive for Cx-43 staining, while LGA tissue samples exhibited moderate staining for Cx-43 and average MI, and GBM biopsies showed significantly lower levels of Cx-43 and high MI. Functional coupling was assayed using fluorescence recovery after photobleach (FRAP). We found that cells from glioma cell lines and primary cultures of human astrocytes from GBM tissues displayed significantly lower degrees of gap junction intercellular communication (GJIC) as indicated by longer and less complete recovery from photobleaching. Mean recovery values were GBM 23.8% +/- 11.4%, LGA 49.4% +/- 47%, and nontumor astrocytes 67.2% +/- 8.4%. Western blot analysis of several human glioma cell lines and tissue biopsies showed variable expression levels of Cx-43, which correlated negatively with the extent of recovery in the same samples. Taken together, our findings suggest that high-grade brain tumors show reduced intercellular communication and a decrease in connexin-43 protein levels.

Aggregation studies of humic acid using multiangle laser light scattering.

An aqueous phase study of the naturally occurring dissolved organic carbon humic acid (HA) is performed using multiangle laser light scattering (MALLS) to measure its RMS radius and average molecular weight. A Zimm plot using Berry Formulism and a second-order fit gave an average molar mass of 1.164 x 10(8) g/mol and an average RMS radius (Rz) of 436.0 +/- 36 nm at 25 degrees C.

Role of lysophosphatidic acid and rho in glioma cell motility.

We have studied the effects of the bioactive phospholipid lysophosphatidic acid (LPA) on cell lines derived from highly invasive human glioblastoma multiforme (GBM). Using transwell migration assays, we show that LPA stimulates both chemokinetic and chemotactic migration of glioma cells. Blood brain barrier breakdown and leakage of serum components that most likely include LPA are common features of GBM. Therefore, the effects of LPA on glioma cell motility are intriguing given the fact that, in vivo, GBM cells often migrate great distances from the main tumor, rendering successful therapy extremely difficult. We show here that LPA initiates a variety of signaling cascades in glioma cells. LPA-enhanced transwell migration was sensitive to pertussis toxin (PTX) treatment suggesting an important role for G(i) subtype of G proteins. LPA also stimulated Ca(2+) fluctuations and activation of extracellular signal-regulated kinases (ERKS) 1 and 2, although blocking either pathway had little effect on glioma cell migration. Exposure of glioma cells to LPA resulted in phosphorylation of the regulatory light chain (RLC) of myosin II and the formation of stress fibers and focal adhesions. These effects were blocked by Y-27632, an inhibitor of Rho-activated ROCK kinases. Time-lapse video microscopy revealed that Y-27632-treatment caused cells to assume long thin morphologies that suggested deficiencies in the contractile apparatus. Furthermore, many cells exhibited a conspicuous extension of processes when Rho/ROCK kinase cascades were inhibited. The above results suggest that LPA/Rho signaling cascades play important roles in glioma cell motility and that exposure of tumor cells to LPA in vivo may contribute to their invasive phenotype.

Recording of intracellular Ca2+, Cl-, pH and membrane potential in cultured astrocytes using a fluorescence plate reader.

A multi-well fluorescence plate reader was used to determine changes in intracellular ionic concentrations and changes in transmembrane voltage in primary cortical or hippocampal astrocytes. Recordings were compared to those obtained using a traditional microscope-based imaging setup that utilizes a monochromatic light source for excitation and an intensified CCD camera for detection. Measurement of pHi with the ratiometric dye BCECF provided resolution similar to that of a microscopic approach. We also demonstrate using Fluo-3 that the measurement of glutamate-induced [Ca2+]i fluctuations are comparable to recordings on the microscope-based system when 25 cells were averaged. This is expected because the plate reader averages responses from many cells. Voltage changes induced by changes in K+(o) from 3 to 5 mM were readily resolvable with the plate reader using the potentiometric dye bis-oxynol, and overall sensitivity was similar to that of microscopically-determined voltage changes. We also found that the plate reader was capable of resolving GABA-induced Cl-(i) fluctuations using the Cl(-)-sensitive indicator MEQ. From these experiments we conclude that multi-well fluorescence plate readers can be used to effectively record changes in intracellular ion concentrations and transmembrane voltage of populations of cells affording time and amplitude resolution approaching that of conventional fluorescence imaging methods. In addition, plate reader-based fluorescence studies demonstrate the added capability to rapidly screen large numbers of samples.

Modulation of glioma cell migration and invasion using Cl(-) and K(+) ion channel blockers.

Human malignant gliomas are highly invasive tumors. Mechanisms that allow glioma cells to disseminate, migrating through the narrow extracellular brain spaces are poorly understood. We recently demonstrated expression of large voltage-dependent chloride (Cl(-)) currents, selectively expressed by human glioma cells in vitro and in situ (Ullrich et al., 1998). Currents are sensitive to several Cl(-) channel blockers, including chlorotoxin (Ctx), (Ullrich and Sontheimer; 1996; Ullrich et al; 1996), tetraethylammonium chloride (TEA), and tamoxifen (Ransom and Sontheimer, 1998). Using Transwell migration assays, we show that blockade of glioma Cl(-) channels specifically inhibits tumor cell migration in a dose-dependent manner. Ctx (5 microM), tamoxifen (10 microM), and TEA (1 mM) also prevented invasion of human glioma cells into fetal rat brain aggregates, used as an in vitro model to assess tumor invasiveness. Anion replacement studies suggest that permeation of chloride ions through glioma chloride channel is obligatory for cell migration. Osmotically induced cell swelling and subsequent regulatory volume decrease (RVD) in cultured glioma cells were reversibly prevented by 1 mM TEA, 10 microM tamoxifen, and irreversibly blocked by 5 microM Ctx added to the hypotonic media. Cl(-) fluxes associated with adaptive shape changes elicited by cell swelling and RVD in glioma cells were inhibited by 5 microM Ctx, 10 microM tamoxifen, and 1 mM TEA, as determined using the Cl(-)-sensitive fluorescent dye 6-methoxy-N-ethylquinolinium iodide. Collectively, these data suggest that chloride channels in glioma cells may enable tumor invasiveness, presumably by facilitating cell shape and cell volume changes that are more conducive to migration and invasion.

Glioma migration can be blocked by nontoxic inhibitors of myosin II.

Anaplastic gliomas are infiltrative tumors, and their ability to migrate through normal brain contributes to their highly malignant behavior. Invasion of brain requires cell motility, which in turn depends on the activity of the cytoskeleton. A cytoskeletal component central to this process is myosin II, the cytoplasmic analogue of smooth and skeletal muscle myosin. Myosin II activity is regulated by the enzyme myosin light chain kinase, which activates myosin II by phosphorylating it on its regulatory light chain. We have investigated the role of myosin II in glioma motility and invasiveness by examining the effects of two inhibitors of myosin light chain kinase, ML7 and KT5926. Both drugs are potent inhibitors of both glioma motility, as measured by a scrape motility assay, and an in vitro haptotaxis assay. The inhibition of in vitro haptotaxis follows the dose-response relationship expected for competitive inhibition of myosin light chain kinase by these drugs and is seen at drug concentrations that are nontoxic. These results highlight the important role that myosin II contributes to glioma invasiveness and suggest that it may serve as a target in future strategies at blocking invasion by these tumors.

Lysophosphatidic acid stimulates actomyosin contraction in astrocytes.

Lysophosphatidic acid (LPA) is an extracellular signaling molecule that can enter the central nervous system following injury or diseases that disrupt the blood-brain-barrier. Using a combination of time-lapse microscopy, immunocytochemistry, and biochemical techniques, we demonstrate that LPA stimulates profound changes in astrocyte morphology that are due to effects on the actomyosin cytoskeleton. Flat astrocytes in primary culture display prominent actin stress fibers. Treatment with the myosin light chain kinase inhibitor, ML-9, causes stress fiber dissolution and dramatic morphology changes including rounding of the cell body and the formation of processes. LPA can stabilize actin stress fibers and inhibit the morphology changes in ML-9-treated cells. Furthermore, this activity is dependent upon activation of the GTP-binding protein Rho as evidenced by the ability of C3 exoenzyme, a specific inhibitor of Rho, to block the effect. Phosphorylation of the regulatory light (RLC) chain initiates conformational changes in myosin II that result in the formation of myosin filaments and the recruitment of actin into contractile stress fibers. LPA-induced stabilization of stress fibers is accompanied by increases in phosphorylation of the RLC of myosin. Furthermore, astrocytes grown on flexible silicone undergo rapid contraction in response to LPA treatment. The forces generated by these cells manifest themselves as increased wrinkling in the silicone. The observed contraction and accompanying increases in regulatory light chain phosphorylation suggest that LPA-induced signaling cascades in astrocytes regulate actin/myosin interactions.

Skin irritation potential of mixed surfactant systems.

Virtually all current detergent formulations contain mixtures of surfactants. Our experience and test data on these formulations, which is in agreement with that of many others, has shown that in use the formulations exhibit lower acute irritation potential than predicted by simple summation of the irritation potential of the individual actives. Using the criteria of the Dangerous Preparations Directive (EC Directive 88/379/EEC), many of these formulations classify as irritant in the neat state, with consequent labelling requirements. Such classification is based on addition of irritant components giving a total concentration which exceeds a nominal threshold. In this study, mixtures of surfactants were tested by application to a panel of 31 human volunteers for up to 4 hr, using the technique established for the assessment of acute skin irritation potential. The positive control, sodium dodecyl sulfate (SDS) at 20% concentration, gave an 84% positive response. Dimethyl dodecyl amido betaine (DDAB) at the same concentration gave a 94% response. However, a combination of 20% of each of these surfactants in the same panellists gave a response of only 44%--a significant reduction in the irritation potential. A further test conducted with a mixture of 10% SDS and 10% DDAB in a second panel gave a 31% positive response compared with a 94% positive response to the 20% SDS control in that panel. These results clearly demonstrate that the acute irritation potential of mixed surfactants cannot be predicted by simple summation of the irritation potential of the component substances. Initial results of the mechanistic investigation indicate that the reduced irritation induced by the mixed surfactant systems correlates with a reduced critical micelle concentration (CMC). However, the reduced CMC itself seems not to be responsible for the lowered irritation, since these experiments were conducted at concentrations well above the CMC. It is proposed that the critical event leading to skin irritation is binding to skin protein and that in mixed surfactant systems, the individual surfactants exhibit less affinity for this protein.

Spontaneous intracellular calcium oscillations in cortical astrocytes from a patient with intractable childhood epilepsy (Rasmussen's encephalitis).

Many studies have demonstrated that astrocytes respond with fluctuations in intracellular calcium concentration ([Ca2+]i) and membrane potential following the application of a number of ligands. Moreover, calcium (Ca2+) waves that spread through astrocytic syncitia have been described in numerous reports. We had the rare opportunity to study Ca2+ responses in astrocytes obtained from a patient diagnosed with Rasmussen's encephalitis, a rare form of intractable epilepsy. Using the ratiometric fluorescent indicator fura-2, we observed large spontaneous [Ca2+]i oscillations. The mean time between initial rise in [Ca2+]i and the return to baseline was 5.1 +/- 0.19 minutes (SEM; n = 201) and [Ca2+]i increased to a mean level of 271 +/- 8 nM (SEM; n = 201) from a baseline of 136 +/- 6 nM (SEM; n = 201). Removal of Ca2+ from the perfusion solution combined with the addition of the Ca2+ chelator EGTA (2 mM) completely but reversibly eliminated all oscillations suggesting the fluctuations were dependent on Ca2+ flux across the membrane. The percentage of cells undergoing spontaneous changes in [Ca2+]i decreased over time in culture. At 10-11 days postsurgery, approximately 70% of the cells were exhibiting this behavior, and by day 23 transients were no longer observed. We did not observe comparable spontaneous [Ca2+]i oscillations in rat cortical astrocytes. The potential that the spontaneous [Ca2+]i oscillations observed may be a unique feature of epileptic tissues is discussed.

Bovine serum albumin and lysophosphatidic acid stimulate calcium mobilization and reversal of cAMP-induced stellation in rat spinal cord astrocytes.

We report that lysophosphatidic acid (LPA) stimulates dynamic calcium (Ca2+) fluctuations and morphological rearrangements in astrocytes derived from neonatal rat spinal cord. Addition of 10 microM LPA elicited single Ca2+ transients, or biphasic oscillations and sustained increases in intracellular Ca2+ ([Ca2+]i). The biphasic Ca2+ response consisted of an initial release from intracellular stores, often followed by a sustained elevation or continued oscillations that required Ca2+ flux across the cell membrane. The type of Ca2+ response, but not the overall magnitude, was dependent on LPA concentration. Higher concentrations (> 10 microM) often elicited sustained increases in [Ca2+]i, while lower concentrations stimulated oscillations or single Ca2+ transients. It has previously been established that agents that elevate cyclic adenosine monophosphate (cAMP) induce flat astrocytes to adopt a more stellate morphology. LPA can completely reverse this morphological change at a half-maximal concentration of 215 nM. Inhibiting LPA-induced [Ca2+]i fluctuations using BAPTA-AM to buffer [Ca2+]i and EGTA in the bath to prevent transmembrane flux had little effect on the ability of LPA to reverse stellation. LPA is found bound to serum albumin, in which crude preparations have been shown to induce various physiological responses in a number of cell types. Many of the activities have been attributed to albumin-associated lipid factors including LPA. We show that lipid factors associated with BSA can mimic the effect of LPA in both Ca2+ mobilization and reversal of cAMP-induced stellation.

Proton magnetic resonance studies of methionine enkephalin.

In the literature there exist two conflicting pieces of data concerning the thermodynamics of the Cu(2+)-methionine enkephalin complex. One study utilized 1H nuclear magnetic resonance (NMR) to study the solution phase Cu(2+)-enkephalin binding and reported a strong interaction over a wide pH range. Another study utilized potentiometry to measure a log beta 101 of 4.6, a value representative of a weak metal-ligand complex. In order to resolve this discrepancy a 300 MHz 1H NMR is used to study the protonation sequence of methionine enkaphalin, a bioactive peptide, and its complexation with seven diamagnetic metals (Ba2+, Ca2+, Zn2+, La2+, Mg2+, Sr2+ and Cd2+).

Effect of heparin infusates in umbilical arterial catheters on frequency of thrombotic complications.

We studied 111 infants requiring an umbilical artery catheter, 59 with heparin and 52 without. Thirty-four thrombi were detected, 16 in the heparin group and 18 in the control group. The numbers of thrombi in the two groups was not significantly different, but the number of clotted or nonfunctioning umbilical artery catheters was greater in the control group (P less than 0.05), as was the incidence of hypertension (P less than 0.05). There were no other significant differences between the two groups. We conclude that the use of low doses of heparin may not change the incidence of umbilical artery catheter-related thrombi, but it does appear to lower the incidence of their sequelae.

Two fatalities resulting from Tessalon (benzonatate).

Two fatal cases involving Tessalon (benzonatate) were quantitated by ultraviolet (UV) and high performance liquid chromatography (HPLC). The first case involved an infant found choking; Tessalon perles were found with the child. The second case involved a successful suicide of an 18-year-old that consumed a "handful" of phenytoin and benzonatate and expired within 1 hr of the ingestion. Blood, brain and kidney concentrations are reported. These are believed to be the first two reported cases involving this compound.

Comparative study of postmortem barbiturates, methadone, and morphine in vitreous humor, blood, and tissue.

With the introduction of radioimmunoassay (RIA) techniques, it has become toxicologically possible to determine drug concentrations in postmortem vitreous humor. This study demonstrates and confirms this toxicological feasibility. In 49 medical examiner's drug related cases, postmortem tissue levels of morphine, barbiturates, and methadone were compared to the vitreous humor.

Methane deaths? Was it the cause?

In the routine performance of their jobs, three men consecutively descended into an open drainage pit to recover a fallen grate lid. Each man, in turn, was immediately overcome and died within minutes of his descent. Initial analysis of the pit's air indicated a methane level of 15%. Therefore, it was initially assumed that death was attributable to methane poisoning. Postmortem analysis of the victims' tissues, however, yielded methane levels in only faint trace quantities (0-100 mcg/100 g range). Analysis of air samples taken at various pit levels revealed that as one descended, there was a decrease in oxygen levels, from 20% at the top to 3% at the bottom. CO2 levels, however, increased from the top of the pit, and reached a level of 22% at the 6-ft. depth of the pit. The accepted lethal level is only 10%. The cause originally attributed to these deaths was shown to be in error. This paper demonstrates the importance of proper investigation of the "scene of occurrence" in order to properly certify the cause of death.

Separation of organophosphate, chlorinated, and carbamate insecticides using conventional and reverse-phase thin-layer chromatography.

This paper demonstrates that the RPTLC system can be used as a confirmation tool for regular adsorption TLC because it separates the pesticides of interest and also reverses their positions on the plate due to their partitioning with water solubility. A clinical procedure was devised for screening samples of gastric and urine for chlorinated, organophosphate, and carbamate insecticides via the use of conventional TLC. The presence of these insecticides was then confirmed by reverse-phase TLC via spotted TLC plates. In view of the plethora of insecticides currently found in the home, and in view of the numerous incidents of adults and children accidentally or purposely ingesting them. This confirming procedure should prove to be a clinically valuable tool in crucial emergency situations.

A study of the myocardial depressant factor and its relative influence in drug/alcohol mortality.

A shock factor, a low molecular weight peptide, has been isolated from postmortem blood. High levels of this peptide, which depresses the myocardium, were seen in cases where drug overdose or alcoholism, or both, were the cause of death. An elevated myocardial depressant factor (MDF) level also demonstrated in a fire victim and a patient in cardiogenic shock. The peptide analysis was accomplished by using an isolated cat papillary muscle followed by paper chromatographic confirmation. Postmortem electrolytes, alcohol, and various toxic agents were eliminated as causes of myocardial depression in the isolated cat papillary muscle assay. The presence of elevated MDF levels may be significant in the overall death process.