Biomedical subjects
Publications and source records attributed to M Zimmer.
ylxL was found to be the last gene of the fla/che operon in Bacillus subtilis and is cotranscribed with the gene for the flagellum-specific alternate sigma factor, sigma(D). The ylxL gene was disrupted by insertional mutagenesis, and the resultant mutant strain was found to be compromised for sigma(D)-dependent functions.
OBJECTIVE: The purpose of this study was to assess whether or not the information on progression of illness and therapeutic interventions included in a computerized SOFA (Sequential Organ Failure Assessment) score and derived measures (maximum SOFA, total maximum SOFA and delta SOFA) influences the correlation with length of stay (LOS) at an operative intensive care unit (ICU). METHODS: During a period of one year 524 patients aging over 18 years who stayed more than 24 hours at an operative ICU were included into this study. Based on SQL scripts (Structured Query Language), a computerized SOFA score and the derived measures "maximum-SOFA", "total-maximum-SOFA" and "delta-SOFA" of all patients were retrospectively calculated for each day at ICU using routine data recorded with the patient data management system ICUData (IMESO GmbH, Hüttenberg, Germany). "Maximum-SOFA" and "total-maximum-SOFA" are measures for the severity of illness or cumulative organ dysfunction. "Delta-SOFA" measures the magnitude of organ dysfunction developing during ICU stay and is therefore potentially influenced by therapeutic measures. Analysis was based on exact LOS in hours. RESULTS: Mean LOS of the whole collective was 5.2 +/- 6.8 days (median 2.8, range 1 - 51). Correlation of LOS depending on score results increased from "admission-SOFA" (r = 0.280), to "maximum-SOFA" (r = 0.444), "total-maximum-SOFA" (r = 0.503), and "delta-SOFA" (r = 0.576). CONCLUSION: Therefore, the sum of information on progression of illness and therapeutic interventions included in derived measures improves the correlation with LOS at an ICU compared to the "admission-SOFA".
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The recently discovered prokaryotic signal transducer HemAT, which has been described in both Archaea and Bacteria, mediates aerotactic responses. The N-terminal regions of HemAT from the archaeon Halobacterium salinarum (HemAT-Hs) and from the Gram-positive bacterium Bacillus subtilis (HemAT-Bs) contain a myoglobin-like motif, display characteristic heme-protein absorption spectra, and bind oxygen reversibly. Recombinant HemAT-Hs and HemAT-Bs shorter than 195 and 176 residues, respectively, do not bind heme effectively. Sequence homology comparisons and three-dimensional modeling predict that His-123 is the proximal heme-binding residue in HemAT from both species. The work described here used site-specific mutagenesis and spectroscopy to confirm this prediction, thereby providing direct evidence for a functional domain of prokaryotic signal transducers that bind heme in a globin fold. We postulate that this domain is part of a globin-coupled sensor (GCS) motif that exists as a two-domain transducer having no similarity to the PER-ARNT-SIM (PAS)-domain superfamily transducers. Using the GCS motif, we have identified several two-domain sensors in a variety of prokaryotes. We have cloned, expressed, and purified two potential globin-coupled sensors and performed spectral analysis on them. Both bind heme and show myoglobin-like spectra. This observation suggests that the general function of GCS-type transducers is to bind diatomic oxygen and perhaps other gaseous ligands, and to transmit a conformational signal through a linked signaling domain.
Density functional calculations using hybrid functionals (B3LYP) have been performed to study the mechanism of the autocatalytic posttranslational cyclization observed in histidine ammonia-lyase. Two mechanisms were analyzed, the commonly accepted mechanism in which cyclization precedes dehydrogenation (reduced mechanism) and a mechanism in which dehydrogenation precedes cyclization (oxidized mechanism). The reduced pathway is not supported by the calculations, while the alternative oxidized mechanism where a dehydration occurs prior to the formation of the ring yields reasonable energetics for the system. Database searches showed that the oxidative mechanism in which the formation of the dehydro amino acids in residue i + 1 precedes the cyclization is also structurally advantageous as it results in shorter distances between the carbonyl carbon of residue i and the amide nitrogen of residue i + 2 and, therefore, preorganizes the protein for cyclization. Conformational searches showed that these distances were also unusually short and exhibited very little variation in the Delta-Ala143 HAL tetramer, indicating that like GFP the tetrameric form of HAL is rigidly preorganized for cyclization. The monomeric form of HAL is less preorganized than the tetrameric form of HAL. Dehydro amino acids aid in the preorganization, but the main driving force in the rigid tight turn formation is the influence of the surrounding protein.
Growing axons follow highly stereotypical pathways, guided by a variety of attractive and repulsive cues, before establishing specific connections with distant targets. A particularly well-known example that illustrates the complexity of axonal migration pathways involves the axonal projections of motor neurons located in the motor cortex. These projections take a complex route during which they first cross the midline, then form the corticospinal tract, and ultimately connect with motor neurons in the contralateral side of the spinal cord. These obligatory contralateral connections account for why one side of the brain controls movement on the opposing side of the body. The netrins and slits provide well-known midline signals that regulate axonal crossings at the midline. Herein we report that a member of the ephrin family, ephrin-B3, also plays a key role at the midline to regulate axonal crossing. In particular, we show that ephrin-B3 acts as the midline barrier that prevents corticospinal tract projections from recrossing when they enter the spinal gray matter. We report that in ephrin-B3(-/-) mice, corticospinal tract projections freely recross in the spinal gray matter, such that the motor cortex on one side of the brain now provides bilateral input to the spinal cord. This neuroanatomical abnormality in ephrin-B3(-/-) mice correlates with loss of unilateral motor control, yielding mice that simultaneously move their right and left limbs and thus have a peculiar hopping gait quite unlike the alternate step gait displayed by normal mice. The corticospinal and walking defects in ephrin-B3(-/-) mice resemble those recently reported for mice lacking the EphA4 receptor, which binds ephrin-B3 as well as other ephrins, suggesting that the binding of EphA4-bearing axonal processes to ephrin-B3 at the midline provides the repulsive signal that prevents corticospinal tract projections from recrossing the midline in the developing spinal cord.
No crystal structure of metallobleomycin (BLM) exists, and the exact coordination mode of the ligand is unknown. To date, spectroscopic investigations of BLM complexes and crystal structures of BLM models have been used to propose its metal coordination sites. This has led to contradictory interpretations of the metal coordination sphere in BLM. Inorganic molecular mechanics and configurational/conformational searches were used to analyze HOO-CoBLM A2, H2O-CoBLM A2, and HOO-CoPEP with commonly proposed binding geometries. The lowest energy binding geometry found was one with the mannose carbamoyl bound to the cobalt ion. The Monte Carlo dihedral and translational variational searches were able to find most of the configurations available to cobalt(III) bleomycin in the three binding geometries examined.
Charcot-Marie-Tooth type 4B (CMT4B), an autosomal recessive demyelinating neuropathy characterized by focally folded myelin sheaths in the peripheral nerve, has been associated with mutations in the gene encoding myotubularin-related protein 2, MTMR2, on chromosome 11q22. To investigate whether mutations in MTMR2 may also cause different forms of CMT, we screened 183 unrelated patients with a broad spectrum of CMT and related neuropathies using denaturing high-performance liquid chromatography. We identified four frequent and three rare exonic variants; two of the rare variants were identified in two unrelated patients with congenital hypomyelinating neuropathy and not in the normal controls. Our results suggest that loss-of-function mutations in MTMR2 are preferentially associated with the CMT4B phenotype.
Green fluorescent protein (GFP) is an extensively used fluorescent tag. Photoisomerization between two spectroscopically distinct states in wild-type GFP is responsible for its two visible absorption bands at 398 nm (A) and 475 nm (B). We have used molecular mechanics and database analysis to support the suggestion of other researchers that the anionic form of the GFP chromophore is responsible for the B absorption band, while the phenol form is responsible for band A. The anionic (-Otyr, Nimid, Glu222H) species is the only form that has a low energy pathway allowing for isomerization of Thr203 to a conformation where it stabilizes the phenolate form and is therefore the most likely species responsible for the B absorption band. The rotation of the Thr203 side-chain is restricted; this may be significant in the formation of the intermediate state which is central to the photoisomerization. Our calculations support the most commonly accepted mechanism for photoisomerization, and we have shown that the 201LSTQS205 sequence does not allow a g+ conformation for Thr203.
For clinical diagnostic routine we developed a fast DNA typing of HLA-B27 by PCR and real-time detection using LightCycler technology. The method combines the sensitivity and specificity of PCR with the swiftness of the LightCycler system. The amplification step was performed with a primer set coding for a region in the third exon common to B*2701 to B*2705. The PCR cycles were monitored continuously using the SYBR Green I dye. Beta-globin was used as an internal control. An analysis of 32 samples with one PCR run was completed within 40 minutes. After amplification a melting curve analysis permitted the accurate identification of the PCR amplicons. The mean melting temperatures (Tm) were 90.5 degrees C and 87.3 degrees C, which are characteristic for HLA-B27 and beta-globin, respectively. A comparison of 300 samples which were typed for HLA-B27 with a conventional sequence-specific polymerase chain reaction (SSP-PCR) and with the new method demonstrated a perfect correlation (specificity 100%). In summary, the method described is fast, reliable, cost-effective and well adapted for routine laboratory testing.
OBJECTIVES AND STUDY DESIGN: The analysis of influence of closure and nonclosure of the visceral peritoneum during abdominal gynecological surgery was done. MATERIALS AND METHODS: A postoperative course and late consequences (occurrence of adhesions) in the 427 patients were studied. There were two groups of patients: I group--203 patients who had visceral peritoneum closed, II group--224 patients who had visceral peritoneum non closed. RESULTS: There were not significant differences in postoperative course in both groups. In the group where the visceral peritoneum was not closed occurrence of the adhesions was significantly decreased, when compared the group where the visceral peritoneum was closed. CONCLUSIONS: 1. Nonclosure of the visceral peritoneum doesn't increase the incidence of early postoperative complications. 2. Nonclosure of the visceral peritoneum during abdominal gynecological surgery decreased the incidence of adhesions. 3. Nonclosure of the visceral peritoneum prevents dislocation and ligation of the ureter.
OBJECTIVES: To estimate the usefulness of hysteroscopy (HSC) and hysterosalpingography (HSG) examination on a retrospective study. DESIGN: Canulisation and morphology of ovarian tube detected during HSG and reactivity of uterine tubal ostia observed in HSC were compared. MATERIAL AND METHODS: The results of HSG and HSC examination performed on 125 young infertile women whom the tubal uterine factor of infertility was suspected were analyzed 250 ovarian tubes, after previous selection were taken into consideration. The analysis had a retrospective character. RESULTS: The analyzed group of women were divided into 3 group which differed from each other in reactivate of uterine ostia of the ovarian tubes. The first group first with correct reactivity of tubal ostia was the largest. The authors observed canulisation, which was the best in the first group and morphologic changes in the internal structure of the ovarian tubes. The highest percentage of these changes were observed in group three with unreactive uterine ostia. CONCLUSIONS: The HSG and HSC examination are supplementary examinations. The use of both methods together increases their diagnostic value and gives a more correct estimation of tubal status.
The muscle-specific receptor tyrosine kinase MuSK plays a crucial role in neuromuscular synapse formation. Activation of MuSK is induced by agrin leading to clustering of several proteins, including acetylcholine receptors, at synaptic sites. In a first step to elucidate the signal transduction cascade following MuSK activation and leading to clustering of synaptic proteins, we sought to identify the tyrosine residues that are phosphorylated in activated MuSK. We mapped the tyrosine residues that are phosphorylated in vitro and in vivo using methods that provide high sensitivity and do not require radioactive tracers. We expressed MuSK in insect cells by using a baculovirus expression vector and mapped the tyrosines that are phosphorylated in MuSK in an in vitro kinase assay using matrix-assisted laser desorption ionization MS to sequence tryptic peptides fractionated by HPLC. In addition, we isolated MuSK from Torpedo electric organ and used nanoelectrospray tandem mass spectrometry and parent ion scanning to identify the tyrosine residues that are phosphorylated in activated, endogenous MuSK in vivo. We found that six of the nineteen intracellular tyrosine residues in MuSK are phosphorylated in activated MuSK: the juxtamembrane tyrosine (Y553), the tyrosines within the activation loop (Y750, Y754, and Y755), a tyrosine near the beginning of the kinase domain (Y576), and a tyrosine (Y812) within the C-terminal lobe of the kinase domain. Our biochemical data are consistent with results from functional experiments and establish a good correlation between tyrosine residues that are phosphorylated in activated MuSK and tyrosines that are required for MuSK signaling.
Haem-containing proteins such as haemoglobin and myoglobin play an essential role in oxygen transport and storage. Comparison of the amino-acid sequences of globins from Bacteria and Eukarya suggests that they share an early common ancestor, even though the proteins perform different functions in these two kingdoms. Until now, no members of the globin family have been found in the third kingdom, Archaea. Recent studies of biological signalling in the Bacteria and Eukarya have revealed a new class of haem-containing proteins that serve as sensors. Until now, no haem-based sensor has been described in the Archaea. Here we report the first myoglobin-like, haem-containing protein in the Archaea, and the first haem-based aerotactic transducer in the Bacteria (termed HemAT-Hs for the archaeon Halobacterium salinarum, and HemAT-Bs for Bacillus subtilis). These proteins exhibit spectral properties similar to those of myoglobin and trigger aerotactic responses.
We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and 3H-inulin. The main ions in the hemolymph were Na+, K+, Mg2+ , Ca2+, and Cl-. The Ca2+ concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca2+ from the cuticle and sternal CaCO3 deposits. The K+ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na+, K+, Mg2+, and Cl- concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater.
Microcin B 17 (MccB17) undergoes an enzyme catalyzed posttranslational modification to form four oxazole and four thiazole rings. Four of these rings form 4,2 - connected biheterocyclic functionalities. In this study, the hexapeptide sequence surrounding the first biheterocyclization site of microcin B17 was examined using computational calculations and database analysis to see if it was preorganized for cyclization in a manner similar to that found in the autocatalytic posttranslational cyclization of Green Fluorescent Protein (GFP). Attention was focused on the intermolecular distances between the sulfur and oxygen atoms of the cysteine and serine residues and the carbonyl carbons which they attack in the ring formation. Conformational searches located some low energy conformations that contained relatively short oxygen to carbonyl carbon distances, which indicated that the oxazole forming fragment in microcin B17 is preorganized for cyclization. However, the lack of any clear patterns for the sulfur to carbon distances show that the side-chain of cysteine does not adopt any low energy conformations that are geometrically preorganized for cyclization. The MccB17 synthetase enzyme complex which catalyzes the cyclization process therefore has both steric and electronic functions. The data obtained in this investigation is in agreement with empirical data which shows that biheterocyclization will only occur if the thiazole forms before the oxazole.
A thorough conformational search of all the conformations available to oxygen-bound urea within wild-type urease was carried out. Identical low energy urea conformations were obtained by a Ramachandran type plot for the NHis272-Ni1-O-Curea, and Ni1-O-Curea-Nurea dihedral angles. Ramachandran plots, with active sites and protonation states modified to model the different urease mechanisms, were used to evaluate the different mechanisms. Based upon the low energy conformations available to urea in the active site of wild-type urease one can conclude that the traditional "His320 acts as a base" mechanism is unlikely. while the N,O urea bridged and the reverse protonation mechanisms cannot be ruled out. A consensus hydrogen-bonding network that does not favor any of the mechanisms has been reconfirmed by the extensive conformational search.
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