Extremophile-inspired strategies for enzymatic biomass saccharification.

Domestic ethanol production in the USA relies on starch feedstocks using a first generation bioprocess. Enzymes that contribute to this industry remain of critical value in new and established markets as commodity additives and for in planta production. A transition to non-food feedstocks is both desirable and essential to enable larger scale production. This objective would relieve dependence on foreign oil and strengthen the national economy. Feedstocks derived from corn stover, wheat straw, perennial grasses and timber require pretreatment to increase the accessibility of the cellulosic and hemicellulosic substrates to commodity enzymes for saccharification, which is followed by fermentation-based conversion of monosaccharides to ethanol. Hot acid pretreatment is the industrial standard method used to achieve deconstruction of lignocellulosic biomass. Therefore, enzymes that tolerate both acid and heat may contribute toward the improvement of lignocellulosic biomass processing. These enzymes are produced naturally by extremely thermophilic microbes, sometimes called extremophiles. This review summarizes information on enzymes from selected (acido)thermophiles that mediate saccharification of alpha- and beta-linked carbohydrates of relevance to biomass processing.

A silica long base tiltmeter with high stability and resolution.

In order to be able to provide valuable data in multiparameter measurement field operations, tiltmeters need to have a noise level better or equal than 10(-9) rad for a period range from a few minutes to a few years and a long term stability ranging from 10(-7) to 10(-8) rad/yr. Tiltmeter measurements should also be as much as possible insensitive to thermal disturbances, by taking great care of the horizontality of the base line tube first. Secondly, thermal responses have been assessed. We also took great care of the coupling of our tiltmeters with the bedrock. We've designed a long base tiltmeter with sensors in silica which has a low dilatation coefficient. The linear variable displacement transducer is based on coil coupling (powered by an alternative voltage). Finally we show the results of two 100 m silica water tube tiltmeters which were installed in a mine in the French Vosges massif in the framework of a hydrology research project. These instruments show a remarkably good stability (6.5x10(-9) rad/month) and a low noise level (of the order of 10(-11) rad). Toroidal and spheroidal free modes of the Earth were observed after the two last major earthquakes on Sumatra.

Peripheral nerve stimulation for the treatment of chronic pain.

The aim of this retrospective study is to evaluate the role of the implanted peripheral nerve stimulator in patients with pain in a peripheral nerve distribution. The current study is the largest in the literature that examines the role of the implantable peripheral nerve stimulator in the chronic pain patient. Our patient sample included 38 patients (with 41 nerve stimulators), consisting of 19 males and 19 females with a mean age of 44 years (SD=11 years). Four groups of etiologic factors were identified; blunt or sharp nerve trauma (14/38), iatrogenic injuries from surgery (9/38), inadvertent injection of a nerve (9/38) and post surgery for entrapment or tumour (8/38). Stimulation was attempted in 45 patients, but an initial trial failed in 4. Mean follow-up time from implantation of the stimulator was 31 months (SD=19 months). Compensation benefit was an issue in 29 cases (76%). Outcome following implantation was assessed based on pain criteria, narcotic usage and return to normal function/ work. Relief from preoperative pain was judged as good (>50% relief) by 23/38 patients (61%). A total of 15 patients reported fair or poor results (39%). Six patients required removal of their stimulators (15%) due to infection or reduction of pain control after an initial good result. A statistically significant decrease in reported pain level was found postoperatively (p<0.05). Workers' compensation patients have equivalent outcomes to non-compensable patients (p>0.05). Eighteen of 38 (47%) patients reported a significant improvement in their activity levels following stimulator implant. In conclusion, over 60% of patients had a significant improvement in their pain and lifestyle following implantation of peripheral nerve stimulators. We therefore conclude that peripheral nerve stimulation can be useful in decreasing pain in well selected patients with severe pain in the distribution of a peripheral nerve.

Long-term outcomes of children with symptomatic congenital cytomegalovirus disease.

OBJECTIVE: To assess long-term outcomes of children with symptomatic congenital cytomegalovirus (CMV) disease detected at birth. STUDY DESIGN: We used Cox regression to assess risk factors for intellectual disability (intelligence quotient <70), sensorineural hearing loss (SNHL; hearing level ⩾25 dB in any audiometric frequency) and vision impairment (best corrected visual acuity >20 or based on ophthalmologist report). RESULTS: Among 76 case-patients followed through median age of 13 (range: 0-27) years, 56 (74%) had SNHL, 31 (43%, n=72) had intellectual disability and 18 (27%, n=66) had vision impairment; 28 (43%, n=65) had intellectual disability and SNHL with/without vision impairment. Microcephaly was significantly associated with each of the three outcomes. Tissue destruction and dysplastic growth on head computed tomography scan at birth was significantly associated with intellectual disability and SNHL. CONCLUSION: Infants with symptomatic congenital CMV disease may develop moderate to severe impairments that were associated with presence of microcephaly and brain abnormalities.

[Translational research in renal cell cancer. Illustrated by the example of the vascular endothelial growth factor pathway]. / Translation molekularer Zusammenhänge in die klinische Anwendung. Dargestellt am Beispiel des Vascular-Endothelial-Growth-Factor- (VEGF-)Pathway.

For patients with metastatic renal cell cancer (RCC), therapeutic options after cytokine failure are rather limited. There is a considerable need to identify new substances for systemic therapy. Due to upregulation after the loss of a functional von Hippel Lindau gene product, the vascular endothelial growth factor (VEGF) pathway is a promising target for a molecular based therapy. Over the last few years, therapeutic agents have been developed which inhibit this pathway at various levels. Here, we provide an overview of the molecular background and currently used drugs which have entered clinical trials in the setting of metastatic RCC disease. Until now, the results from early clinical trials are very promising, however, the best schedule, dosage, potential combination regimens, as well as long time efficacy, are still to be determined.

Immunochemical identification of a tRNA-independent cytokinin-like compound in Salmonella typhimurium.

Chemical and immunological characterization of Salmonella typhimurium cell extracts indicates that this organism produces a molecule which closely resembles the plant growth regulator, cytokinin. Alcohol-soluble cationic ultraviolet-absorbing material was fractionated by reverse-phase HPLC using gradient conditions optimized previously for modified nucleoside separation. A single hydrophobic compound was identified in the cytokinin region of the gradient, and limited quantities of the compound were prepared by HPLC fractionation of crude extracts. The compound demonstrated significant activity in a radioimmunoassay for cytokinins which detects N6-isopentenylated adenine derivatives. Boronate affinity chromatography indicated the compound is likely to be ribosylated and therefore a nucleoside. These and other tests indicate the compound has the most notable structural characteristics of a cytokinin. Spectral analysis and chromatographic comparison with cytokinin standards indicate the compound also has some unique structural features. Presence of the compound in extracts of an S. typhimurium mutant blocked for synthesis of tRNA-derived cytokinins excluded tRNA as a source for the compound and implicates existence of a tRNA-independent pathway for cytokinin biosynthesis in this bacterial species.

Extragenic pleiotropic mutations that repress glycosyl hydrolase expression in the hyperthermophilic archaeon Sulfolobus solfataricus.

The hyperthermophilic archaeon Sulfolobus solfataricus employs a catabolite repression-like regulatory system to control enzymes involved in carbon and energy metabolism. To better understand the basis of this system, spontaneous glycosyl hydrolase mutants were isolated using a genetic screen for mutations, which reduced expression of the lacS gene. The specific activities of three glycosyl hydrolases, including an alpha-glucosidase (malA), a beta-glycosidase (lacS), and the major secreted alpha-amylase, were measured in the mutant strains using enzyme activity assays, Western blot analysis, and Northern blot analysis. On the basis of these results the mutants were divided into two classes. Group I mutants exhibited a pleiotropic defect in glycosyl hydrolase expression, while a single group II mutant was altered only in lacS expression. PCR, Southern blot analysis, comparative heterologous expression in Escherichia coli, and DNA sequence analysis excluded cis-acting mutations as the explanation for reduced lacS expression in group I mutants. In contrast lacS and flanking sequences were deleted in the group II mutant. Revertants were isolated from group I mutants using a lacS-specific screen and selection. These revertants were pleiotropic and restored glycosyl hydrolase activity either partially or completely to wild-type levels as indicated by enzyme assays and Western blots. The lacS mutation in the group II mutant, however, was nonrevertible. The existence of group I mutants and their revertants reveals the presence of a trans-acting transcriptional regulatory system for glycosyl hydrolase expression.

Use of M13mp phages to study gene regulation, structure and function: cloning and recombinational analysis of genes of the Salmonella typhimurium histidine operon.

A restriction map was determined for a phi 80 lambda dhis transducing phage DNA carrying the Salmonella typhimurium histidine operon. DNA fragments containing the promoter/regulatory region and the first two structural genes of the histidine operon (hisOGD) were identified by their ability to direct regulated synthesis of histidinol dehydrogenase (product of hisD) in a coupled in vitro protein synthesizing system. A 3.1-kb SalI-EcoRI restriction fragment containing the hisOGD region, was subcloned into phage M13mp8 and M13mp9 RF DNAs. Methods are described for shuttling mutant and wild-type bacterial DNA sequences between the M13mp::his phage and host bacterial genomes. Of novel importance is the use of the phage M13 gene II amber mutation to obtain integration of the M13mp::his phage genome into the homologous his region of the bacterial chromosome following transduction of recipients lacking an amber suppressor. This method can be used to facilitate allele replacement with genes carried on M13 transducing phages.

Piperazinylalkyl heterocycles as potential antipsychotic agents.

We recently reported on a series of pyrrole Mannich bases orally active in inhibiting the conditioned avoidance response (CAR) in rats. These compounds exhibit affinity for both D2 and 5-HT1A receptors, and some are noncataleptogenic. Such a profile suggests that they may be potential antipsychotic agents which lack the propensity for causing extrapyramidal side effects and tardive dyskinesias in humans. One of these compounds, 1-[[1-methyl-5-[[4-[2-(1-methylethoxy)phenyl]- 1-piperazinyl]methyl]-1H-pyrrol-2-yl]methyl]-2-piperidinone (RWJ 25730, 1), was chosen for further development but found to be unstable in dilute acid. In order to improve stability, we replaced the pyrrole methylene linkage to the piperazine ring with ethylene, employed ethylene and dicarbonyl as linkers between the lactam and the pyrrole ring, placed electron-withdrawing groups on the pyrrole ring, and substituted acyclic amide for lactam. In addition, we replaced the pyrrole segment with other heterocycles including thiophene, furan, isoxazole, isoxazoline, and pyridine. Generally, replacement of the N-methylpyrrole segment with thiophene, furan, isoxazoline, or pyridine afforded compounds equipotent with 1 in CAR, which were more stable in dilute acid. In the case of side chain or lactam modifications, CAR activity was significantly decreased or abolished, with the exception of 6. For the most part, the modifications to 1 resulted in the decrease or loss of D2 receptor binding. However, within this series, 5-HT1A receptor binding was greatly increased, with thiophene 40 exhibiting an IC50 of 0.07 nM. The CAR activities of pyrroles 6 and 12, thiophene 40, furans 44-47, isoxazolines 49 and 50, and pyridine 54 coupled with their weak or nonexistent D2 binding and strong 5-HT1A binding suggest that they may be acting via a nondopaminergic mechanism or that dopaminergic active metabolites are responsible. Pyrrole 6 and furans 44 and 47 show promise as antipsychotic agents based on their CAR activity, receptor-binding profile, and solution stability.

Effects of smoking and inhalation of carbon monoxide on systolic time intervals and blood pressure. Differences between two types of cigarettes and a cigar.

The effects of smoking and inhalation of carbon monoxide on the systolic time intervals and blood pressure were examined in ten healthy smokers with a mean age of 24.3 years. Each subject smoked a low-nicotine cigarette with a ventilated filter (0.1 mg nicotine, 1.1 vol percent CO), and a high-nicotine plain cigarette (2.6 mg nicotine, 4.5 vol percent CO), as well as a cigar in random sequence and in a standardized way. Cigar smoke was not inhaled. The product heart rate x blood pressure was increased and the left ventricular ejection time index (LVETc) prolonged following smoking the high-nicotine cigarette, whereas changes after smoking the low-nicotine cigarette and the cigar were not as pronounced. These changes are presumably caused by nicotine-induced catecholamine release. Inhalation of CO did not affect cardiac performance acutely as shown by unchanged systolic time intervals. When a high-nicotine cigarette was smoked after the subject received a beta blocker, a significant prolongation of the pre-ejection period index (PEPc) occurred as a result of the increased afterload. Thus, the effects of catecholamines on parameters of myocardial contractility (PEPc, PEP/LVET) were presumably offset by the increased afterload. We conclude that the acute hemodynamic changes of smoking in healthy subjects depend upon the amount of nicotine absorbed.

Nucleotide sequence analysis and heterologous expression of the Erysipelothrix rhusiopathiae dnaJ gene.

DNA sequence analysis of chromosomal DNA from the Gram-positive facultative intracellular pathogen, Erysipelothrix rhusiopathiae has identified a dnaJ heat shock gene homolog. A 1109-bp open reading frame encoding dnaJ is located immediately 3' to the E. rhusiopathiae dnaK gene. The deduced DnaJ amino acid sequence exhibits the modular structure of other members of the DnaJ protein class including a glycine-rich region and the repeating consensus sequence CXXCXGXGX. Heterologous expression of the dnaJ sequence in Escherichia coli resulted in accumulation of a unique 38.9-kDa protein with an isoelectric point of 8.0. Deletion analysis of the dnaJ gene was used to confirm that the overproduced protein was encoded by the dnaJ sequence.

The role of anion channels in osmotically activated taurine release from embryonic skate (Raja eglanteria) heart

Taurine, a major osmolyte of vertebrate hearts, is released from the skate heart at increased rates during hypotonic stress. We tested the hypothesis that this taurine release is mediated by chloride channels activated by swelling. Two inhibitors of the channels, NPPB and DIDS, inhibited the volume-activated release of taurine from embryonic skate hearts. These results support the hypothesis that swelling-activated chloride channels mediate the release of cardiac taurine.

Interaction between naloxone and serotonin in the control of the cardiovascular system in hemorrhaged cats.

We investigated the mechanism for the pressor effect of intravenous administration of naloxone in pentobarbital-anesthetized cats. Comparisons were made between groups of hemorrhaged animals that received either naloxone or an equivalent volume of saline after 1 h of hemorrhage. Two other groups of hemorrhaged animals were depleted of serotonin by pretreatment with para-chlorophenylalanine 40-48 h before the experiment. One group of serotonin-depleted animals received naloxone after 60 min of hemorrhage and the other group received saline. Animals with normal brain serotonin content showed a significant pressor effect following naloxone when compared with animals given saline. Animals with reduced brain serotonin content also had a pressor response following naloxone administration. Serotonin-depleted animals showed an increase in maximum left ventricular dP/dt following naloxone administration when compared to serotonin-depleted animals given saline. Our data are consistent with the hypothesis that naloxone can exert a pressor effect in hemorrhaged cats by actions at central and at peripheral sites. In cats with normal serotonin values, the peripheral action of naloxone is predominant. In serotonin-depleted animals given naloxone, central and peripheral sites contribute to the pressor effect.

Osteochondroma with spinal cord compression: report of three cases.

Multiple osteochondromatosis is the most common of the bone dysplasias, but neurological complications are rare. Osteochondromas of the spine usually arise from the neural arch. Tomography and myelography are useful in determining the site of origin and the relationship to the spinal cord. The tumor is benign, and surgical removal produces an excellent clinical result. Three cases are described illustrating the clinical and radiological manifestations of these tumors.

Improved homogenization of recombinant Escherichia coli following pretreatment with guanidine hydrochloride.

Pretreatment of recombinant Escherichia coli, expressing human growth hormone inclusion bodies, with guanidine hydrochloride and Triton X-100 prior to high-pressure homogenization has been investigated. Homogenates were analyzed for protein release, viscosity, and particle size. We were able to reduce the number of passes required for cell disruption and the number of downstream processing steps required for the recovery of protein from inclusion bodies by pretreating cells with guanidine HCl and Triton X-100. Pretreatment of exponential growth phase cells with 1.5 M guanidine HCl and 1.5% Triton X-100 gave adequate disruption after one pass at 41 MPa with a particle size distribution similar to that for untreated cells disrupted after one pass at 62 MPa. This combination of guanidine HCl and Triton X-100 was also selected so as to wash the inclusion bodies without solubilization of the human growth hormone. Pretreatment of cells with 4 M guanidine HCl produced cell debris that was substantially smaller than the debris from untreated cells and partially solubilized the inclusion bodies. Cells harvested in the stationary growth phase were more resistant to high-pressure homogenization and pretreatment.

Chronic intracranial hypotension.

Acute intracranial hypotension can occur following lumbar puncture or a fall, and sometimes spontaneously. Most cases resolve within weeks or months but some require surgical repair of the defect causing leakage of cerebrospinal fluid (CSF). It is conceivable that such leaks could become chronic if the defect is incompletely sealed. We report the case of a 49-year-old male who presented with a 10-month history of headache associated with a leaking thoracic extradural arachnoid cyst. After this was repaired he reported relief not only of his recent headaches but also of chronic alcohol-related headaches. A long-standing anaemia resolved and tinnitus hyperacusis improved. It is suggested that an injury 30 years before may have initiated the leak of CSF resulting in chronic intracranial hypotension.

Quercetin cumulatively enhances copper induction of metallothionein in intestinal cells.

Wilson's disease, a genetic copper-overload condition, is currently treated with zinc because of the ability of zinc to induce metallothionein. We are interested in nonmetal chemicals that may alter intestinal copper metabolism and thus help to alleviate copper toxicity. Previously, we have shown that quercetin, a dietary flavonoid, can chelate copper. This study further examined the interaction of quercetin and copper in intestinal epithelial cells. We found that quercetin enhanced metallothoinein induction by copper and the effect was dose dependent. Quercetin also exerted a cumulative effect after repeated exposure. Repeated low-dose treatment (3-10 microM) of cells with quercetin can lead to the same effect on metallothoinein as one higher concentration treatment (100 microM). This property of quercetin is distinct from its chemical interaction with copper, but both can contribute to a reduction of copper toxicity. Among other flavonoids tested, two other copper chelators, catechin and rutin, did not increase copper induction of metallothionein, whereas genistein, an isoflavone that does not interact with copper chemically, increased copper induction of metallothionein. The effect of quercetin on copper metabolism is unique. Quercetin decreased zinc-stimulated metallothionein expression and had no effect on the cadmium induction of metallothionein. The clinical application of our observation needs to be explored.

Spontaneous recovery of selective IgA deficiency. Additional case reports and a review.

Patients with selective IgA deficiency, the most common primary immunodeficiency, occasionally may undergo spontaneous recovery. In this paper we present three additional children whose IgA levels spontaneously returned to normal. All three presented with gastrointestinal symptoms, and one child also had frequent upper respiratory infections. We review other cases of spontaneous IgA deficiency, and the role of exogenous function in its etiology, and possible mechanisms of recovery.

Whole-limb irradiation of the lower calf using a six-field electron technique.

The present work demonstrates utilization of electron beam irradiation for the treatment of Kaposi's sarcoma when the full circumference of the lower calf is involved, and when the deep lymphatics are negative for disease. The finite penetration of the electron beam spares deep tissue, preventing the edema associated with photon total limb irradiation. The number of fields with fall-off required to produce a uniform dose to a cylindrical anatomic structure was studied by calculating dose distributions resulting from two-, four-, and six-field techniques for a 5-MeV electron beam and a 9 cm diameter cylinder. The dosimetry and set-up for the six-field technique is demonstrated by a case study. Results show that a six-field electron technique produced a sufficiently uniform dose while remaining relatively easy to set up and use to deliver patient treatment. For the patient case study, dose distributions for the six-field technique showed that (1) the penetration of the 90% dose decreased from 1.5 cm for a single field to approximately 1.0 cm for a 5-MeV beam; (2) the surface dose increased from approximately 70% to 100%, (3) the dose around the circumference of the leg at the depth of 1 cm or less varied from approximately 90% to 120% of the prescribed dose; and (4) the prescribed dose was 2.5 times the maximum central-axis dose from a single field. The six-field treatment was relatively simple to apply and produced an acceptable dose distribution for treatment of Kaposi's sarcoma of the lower calf. This treatment should be applicable to other sites such as the thigh and arms and for other cutaneous diseases such as melanoma.

DnaK-mediated alterations in human growth hormone protein inclusion bodies.

Protein overproduction in microbes frequently results in protein misfolding and aggregation though the molecular basis for this process is unclear. The HSP70 chaperonin, DnaK, was identified as an important factor controlling heterologous protein aggregation in Escherichia coli. Co-overproduction of DnaK significantly reduced human growth hormone (HGH) protein inclusion body formation and the extent of HGH aggregation.