Type VI secretion requires a dynamic contractile phage tail-like structure.

Type VI secretion systems are bacterial virulence-associated nanomachines composed of proteins that are evolutionarily related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.

Initiation of H1-T6SS dueling between Pseudomonas aeruginosa.

The Type VI secretion system (T6SS) is a multicomponent apparatus, present in many Gram-negative bacteria, which can inhibit bacterial prey in various ecological niches. Pseudomonas aeruginosa assembles one of its three T6SS (H1-T6SS) to respond to attacks from adjacent competing bacteria. Surprisingly, repeated assemblies of the H1-T6SS, termed dueling, were described in a monoculture in the absence of an attacker strain; however, the underlying mechanism was unknown. Here, we explored the role of H2-T6SS of P. aeruginosa in triggering H1-T6SS assembly. We show that H2-T6SS inactivation in P. aeruginosa causes a significant reduction in H1-T6SS dueling and that H2-T6SS activity directly triggers retaliation by the H1-T6SS. Intraspecific competition experiments revealed that elimination of H2-T6SS in non-immune prey cells conferred protection from H1-T6SS. Moreover, we show that the H1-T6SS response is triggered independently of the characterized lipase effectors of the H2-T6SS, as well as those of Acinetobacter baylyi and Vibrio cholerae. Our results suggest that H1-T6SS response to H2-T6SS in P. aeruginosa can impact intraspecific competition, particularly when the H1-T6SS effector-immunity pairs differ between strains, and could determine the outcome of multistrain colonization.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa harbors three different Type VI secretion systems (H1, H2, and H3-T6SS), which can translocate toxins that can inhibit bacterial competitors or inflict damage to eukaryotic host cells. Unlike the unregulated T6SS assembly in other Gram-negative bacteria, the H1-T6SS in P. aeruginosa is precisely assembled as a response to various cell damaging attacks from neighboring bacterial cells. Surprisingly, it was observed that neighboring P. aeruginosa cells repeatedly assemble their H1-T6SS toward each other. Mechanisms triggering this "dueling" behavior between sister cells were unknown. In this report, we used a combination of microscopy, genetic and intraspecific competition experiments to show that H2-T6SS initiates H1-T6SS dueling. Our study highlights the interplay between different T6SS clusters in P. aeruginosa, which may influence the outcomes of multistrain competition in various ecological settings such as biofilm formation and colonization of cystic fibrosis lungs.

Enzyme kinetic and substrate-binding studies of the thyroxine to 3,5,3'-triiodothyronine converting enzyme in the rat liver microsomal fraction.

The conversion of T4 to T3 was studied in the rat liver microsomal fraction. A mean Vmax of 0.11 pmol T3 produced per mg microsomal protein/min and a mean apparent Km of 2.1 muM T4 were found. An approximation to the real Km for the experimental conditions used was obtained by applying free instead of total T4 as the substrate concentration. Thus, the Km was found to be 9.7 nM free T4, and changes of Km with different amounts of microsomal protein added were not detected. rT3 was found to be a competitive inhibitor of the T4 to T3 conversion, with a mean apparent Ki of 9.4 nM rT3. Binding studies showed that T4 is bound not only nonspecifically but also to two different classes of specific binding sites. The dissociation constants were 7.5 and 1700 nM t4, and the maximal binding capacities were 58 and 4300 pmol T4/mg microsomal protein, respectively, rT3 and T3 both had one specific binding site besides their unspecific binding to the microsomal fraction. The dissociation constants were found to be 45 nM rT3 and 850 nM T3, respectively; the maximal binding capacities amounted to 75 pmol rT3 and 4600 pmol T3 per mg microsomal protein, respectively. rT3 competes with T4 for its first (apparent Ki, 55 nM rT3) and T3 competes with T4 for its second specific binding site (apparent Ki, 960 nM T3). It is suggested, that the first specific binding site for T4 and the specific binding site for rT3 are identical, and that they represent the 5'-deiodinase. The rT3-induced inhibition of the T4 to T3 conversion is caused by a competition for the binding site of the enzyme. A competition for cofactors may play an additional role. T3 competes with T4 for a different specific binding site, which may contain the 5-deiodinase.

Polymorphism of the human phosphoglycolate phosphatase in Northrhine-Westphalia (F.R.G.) and its application to paternity testing.

Blood samples from 563 unrelated German and from 110 Turkish individuals living in the Düsseldorf area were studied for phosphoglycolate phosphatase polymorphism. The distribution of the observed phenotypes in the population genetic study did not diverge from the expected values according to Hardy-Weinberg law. In our series, the gene frequencies were calculated as follows: (a) Germans: PGP1 = 0.851, PGP2 = 0.118, PGP3 = 0.031, (b) Turks: PGP1 = 0.973, PGP2 = 0.018, PGP3 = 0.009. The assumed autosomal codominant mode of inheritance was confirmed by the examination of 109 mother-child pairs and by analysis of 70 cases of disputed paternity. The plausibility to exclude German non-fathers from paternity is 12.78%.

Further data on the development of red blood cell antigens Lua, Lub, and Cob.

One hundred and fifty-five cord cells were tested for the red blood cell antigens Lua, Lub and Cob in order to collect data on the early postnatal expression of these markers. Additionally, dosage studies were carried out in 8 - 10-month-old heterozygous children. Antigens Lua and Lub revealed to be significantly less expressed in children of both ages compared with those of their mothers, whereas no such differences could be demonstrated in the expression of the antigen Cob.

Type 6 secretion dynamics within and between bacterial cells.

The bacterial type 6 secretion system (T6SS) functions as a virulence factor capable of attacking both eukaryotic and prokaryotic target cells by a process that involves protein transport through a contractile bacteriophage tail-like structure. The T6SS apparatus is composed, in part, of an exterior sheath wrapped around an interior tube. Here, we report that in living cells the cytoplasmic adenosine triphosphatase called ClpV specifically recognizes the contracted T6SS sheath structure, causing its disassembly within seconds. ClpV imaging allowed spatial and temporal documentation of cell-cell interactions (termed T6SS dueling) that likely mark the location of repeated T6SS-mediated protein translocation events between bacterial cells.

How positive and negative expectations shape the experience of visceral pain: an experimental pilot study in healthy women.

BACKGROUND: In order to elucidate placebo and nocebo effects in visceral pain, we analyzed the effects of positive and negative expectations on rectal pain perception, rectal pain thresholds, state anxiety and cortisol responses in healthy women. METHODS: Painful rectal distensions were delivered at baseline, following application of an inert substance combined with either positive instructions of pain relief (placebo group, N = 15), negative instructions of pain increase (nocebo group, N = 17), or neutral instructions (control, N = 15). Perceived pain intensity, unpleasantness/aversion and urge-to-defecate, state anxiety and serum cortisol were determined at baseline, immediately following group-specific instructions and on a second study day after the same instructions (test day). Rectal pain thresholds were determined at baseline and on the test day. KEY RESULTS: Whereas perceived pain intensity was significantly decreased in the placebo group, the nocebo group revealed significantly increased pain intensity ratings, along with significantly greater anticipatory anxiety on the test day (all P < 0.05 vs controls). Cortisol concentrations were significantly increased in the nocebo group following treatment but not on the test day. CONCLUSIONS & INFERENCES: The experience of abdominal pain can be experimentally increased or decreased by inducing positive or negative expectations. Nocebo effects involve a psychological stress response, characterized by increased anticipatory anxiety. These findings further underscore the role of cognitive and emotional factors in the experience of visceral pain, which has implications for the pathophysiology and treatment of patients with chronic abdominal complaints.

Evidence for a "new" allele at the esterase D (E.C. 3.1.1.1.) locus.

An apparently new EsD gene product (EsD*Düsseldorf) was detected by use of horizontal agarose gel electrophoresis (AGE), starch gel electrophoresis (SGE), and isoelectric focusing (IEF). The observed phenotype EsD (1-Düsseldorf) can be distinguished from any known EsD type.

[Unusual Gm-haplotypes possibly due to crossing over]. / Uber ungewöhnliche Gm-Haplotypen, die vermutlich durch Crossing-over entstanden sind.

Four cases are introduced with unusual Gm-haplotypes. Their possible origin is discussed as being either intra- and intergenic or unequal cross over. The last case shows deletions of either y1 or y3 -cistrons due to unequal cross over.

The forensic application of the blood group antigens Lua and Lub.

Blood samples from 507 unrelated persons in Northrhine-Westphalia and from 254 paternity cases were tested for the Lutheran blood group antigens Lua and Lub. The gene frequencies were found to be 0.03 (= Lua) and 0.969 (= Lub).

On recent observations in the rhesus blood group system. I. A weak expression of the Rh antigen e. II. Finding of the rare Rh antigen Cx.

Upon testing blood samples for medico-legal purpose, an exceptionally weak expression of the Rhesus antigen e was observed. There is evidence for the phenotype Rh2rh which is rather unusual in white people. Apart from this, the low incidence antigen Cx has been found in two cases. Its mode of reaction with anti-C and anti-c sera is described.

An evaluation of two concentrations of hyaluronidase for supplementation of peribulbar anaesthesia.

We studied the effect of the addition of hyaluronidase to a mixture of lignocaine 2% and bupivacaine 0.75% for peribulbar anaesthesia in 60 patients presenting for elective intra-ocular surgery. Using a randomised, double-blind design, patients were allocated to one of three groups: no hyaluronidase (group A) (n = 20); hyaluronidase 50 iu.ml-1 (group B) (n = 20); hyaluronidase 150 iu.ml-1 (group C) (n = 20). The speed of onset of the block, the presence of akinesia, analgesia and the need for supplementary injections were assessed. The addition of 150 iu.ml-1 of hyaluronidase resulted in a mean (SEM) time to akinesia of 9.2 (0.9) min compared to 10.9 (0.9) min in the control group and 10.7 (1.1) min in those receiving the lower dose. Fewer patients in group C required a further injection (20%) than those in groups A (40%) or B (45%). None of the findings were statistically significant. Hyaluronidase was not associated with any complications.

[Vitamin B6-sensitive hereditary sideroblastic anemia]. / Vitamin-B6-sensible hereditäre sideroblastische Anämie.

HISTORY: A 30-year-old patient was admitted for investigation of microcytotic, hypochromic anemia (hemoglobin 8.3 g/dl) with splenomegaly. INVESTIGATIONS: Bone marrow smear showed a normocellular marrow with augmented severely dysplastic erythropoesis. Prussian-blue staining revealed an increased number of ring sideroblasts, thus myelodysplastic syndrome (refractory anemia with ringsideroblasts) was suspected. DIAGNOSIS AND TREATMENT: Review of former laboratory values and investigations of the patient's family revealed the correct diagnosis of x-linked sideroblastic anemia (XLSA). The patient was treated with oral pyridoxine. Hemoglobin levels steadily increased, so the diagnosis of pyridoxine-responsive sideroblastic anemia was made. Liver biopsy showed secondary fibrosis with beginning cirrhosis due to iron overload. Therapy with deferoxamine and phlebotomies was initiated. CONCLUSION: XLSA is a rare differential diagnosis of acquired forms of sideroblastic anemias. A high degree of clinical suspicion is necessary for diagnosis because morphological studies such as histology and cytology may not yield conclusive results. A correct diagnosis is especially important because of the uncomplicated therapeutic options.

PGM1a subtyping in Western Germany (Düsseldorf region).

Phosphoglucomutase (PGM1a) subtypes were determined by isoelectric focusing on samples of 496 unrelated individuals. Ten phenotypes were observed as gene products of four alleles at the PGM1 locus, with the following gene frequencies: PGM1a1 = 0.631, PGM1a2 = 0.194, PGM1a3 = 0.126, and PGM1a4 = 0.049. A rare phenotype PGM1(8-a2) was observed.

Preparation of parenchymal and non-parenchymal cells from adult human liver--morphological and biochemical characteristics.

By perfusion of the isolated human liver with collagenase and hyaluronidase a mixed suspension of various cell types was obtained. Pure parenchymal cells were prepared by differential centrifugation, pure non-parenchymal cells by the use of pronase and subsequent isopycnic centrifugation on metrizamide gradients (50-300 g/l). About 90% of the parenchymal and non-parenchymal cells were viable as judged by trypan blue staining. Non-parenchymal cells were not capable fo gluconeogenesis but at high rates. Parenchymal cells retained their ability to form glucose and to accumulate glycogen from fructose greater than lactate/pyruvate greater than alanine. Studies on binding of 125I-labelled insulin by isolated parenchymal cells were performed at 30 degrees C. The binding data may fit with a minimum of two classes of binding sites: (a) high affinity--low capacity sties (Kd approximately 6.6 nmol/l, capacity approximately 16 000 insulin molecules per cell) and (b) low affinity-high capacity sites (Kd approsimately 0.37 mumol/l, capacity approximately 646 000 molecules per cell).

[Isolation of intact liver parenchymal cells by a modified enzymatic method]. / Isolierung intakter Leberparenchymzellen durch eine modifizierte enzymatische Methode

An enzymatic method is described for isolating intact parenchymal cells from rat livers. 3--4 g cells (wet weight) could be isolated from livers of rats weighing 180--230 g. After an in vitro preperfusion of 15 minutes with a Ca-free buffer, collagenase (200 mg/1) and calcium chloride (5.2 mmol/1) were added. Perfusion was continued for another 15 minutes at 37 degrees C. Micromorphological integrity of cell membranes was demonstrated by scanning electron microscopy. With regard to rates of gluconeogenesis and protein synthesis, parenchymal cells isolated according to our method were found to be superior to liver slices and cells isolated by other methods. Ratios of ATP/ADP (5.69) and of lactate/pyruvate (8.64) as parameters of the energetic situation and the redox state resp. were found within the physiological range. Integrity of cell surface receptors was proved by their sensitivity to epinephrine, glucagon and insulin. Glucagon (0.3 mumol/1) and epinephrine (1 mumol/1) and reduced glycogen deposition in hepatocytes of fasted rats by 84.9 % and 95.9 % resp. Both hormones stimulated glycogenolysis in parenchymal cells of fed rats to a similar extent. Urea synthesis was stimulated 29.5 % by glucagon (1 mumol/1), and inhibited 28.0 % by insulin (10 nmol/1). The stimulatory effect of glucagon (1 mumol/1) was abolished by insulin (10 nmol/1).