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Fig4 is a phosphoinositide phosphatase that converts PI3,5P2 to PI3P. Paradoxically, mutation of Fig4 results in lower PI3,5P2, indicating that Fig4 is also required for PI3,5P2 production. Fig4 promotes elevation of PI3,5P2, in part, through stabilization of a protein complex that includes its opposing lipid kinase, Fab1, and the scaffold protein Vac14. Here we show that multiple regions of Fig4 contribute to its roles in the elevation of PI3,5P2: its catalytic site, an N-terminal disease-related surface, and a C-terminal region. We show that mutation of the Fig4 catalytic site enhances the formation of the Fab1-Vac14-Fig4 complex, and reduces the ability to elevate PI3,5P2. This suggests that independent of its lipid phosphatase function, the active site plays a role in the Fab1-Vac14-Fig4 complex. We also show that the N-terminal disease-related surface contributes to the elevation of PI3,5P2 and promotes Fig4 association with Vac14 in a manner that requires the Fig4 C-terminus. We find that the Fig4 C-terminus alone interacts with Vac14 in vivo and retains some functions of full-length Fig4. Thus, a subset of Fig4 functions are independent of its phosphatase domain and at least three regions of Fig4 play roles in the function of the Fab1-Vac14-Fig4 complex.

In most eukaryotes, phosphoinositides (PIs) have crucial roles in multiple cellular functions. Although the cellular levels of phosphatidylinositol 5-phosphate (PI5P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) are extremely low relative to some other PIs, emerging evidence demonstrates that both lipids are crucial for the endocytic pathway, intracellular signaling, and adaptation to stress. Mutations that causes defects in the biosynthesis of PI5P and PI(3,5)P2 are linked to human diseases including neurodegenerative disorders. Here, we review recent findings on cellular roles of PI5P and PI(3,5)P2, as well as the pathophysiological importance of these lipids.Key words: Phosphoinositides, Membrane trafficking, Endocytosis, Vacuoles/Lysosomes, Fab1/PIKfyve.

When choosing between two options, a sensible strategy is to choose the highest value option. To do this, both options must be evaluated and compared. The way value is judged and utility is perceived therefore has strong effects on which option is chosen. Traditionally, value was considered absolute. However, research on human decision-making suggests that, for us, utility is relative and based on past experiences or expectations. The study of successive contrast effects suggests that the same might be true for animals. Here we show that ants which had previously experienced a low quality food source showed higher acceptance of medium quality food (e.g. 0.1M then 0.5M; positive contrast) than if they had received the medium food all along (e.g. 0.5M then 0.5M; control). Ants also showed lower acceptance of medium food when previously offered high quality food (e.g. 2M then 0.5M; negative contrast). Further experiments demonstrate that these contrast effects arise from psychological, not physiological or psychophysical, causes. Pheromone deposition also correlates with perceived reward value, and ants also showed successive contrasts in their pheromone deposition. Contrast effects occurred not only when ants collected private information outside the nest, but also when information was received through trophallactic interactions in the nest. Relative value perception can therefore be expected to have strong effects not only on individual behaviour, but also on collective decision-making.

In most eukaryotes, phosphoinositides (PIs) have crucial roles in multiple cellular functions. Although the cellular levels of phosphatidylinositol 5-phosphate (PI5P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) are extremely low relative to some other PIs, emerging evidence demonstrates that both lipids are crucial for the endocytic pathway, intracellular signaling, and adaptation to stress. Mutations that causes defects in the biosynthesis of PI5P and PI(3,5)P2 are linked to human diseases including neurodegenerative disorders. Here, we review recent findings on cellular roles of PI5P and PI(3,5)P2, as well as the pathophysiological importance of these lipids.

From 2002 to 2013, bovine tuberculosis (bTB) caused by Mycobacterium bovis (M. bovis) has been detected on numerous dairies in California. In total, twelve herds had bTB detected and are included in the case series which describes these recent outbreaks and discusses potential pathways of introduction. Epidemiological investigations to determine the initial source of bTB in each herd included obtaining data on likely pathways of pathogen introduction. Pathways included purchasing cattle, use of heifer-raising operations, commingling of cattle at greater risk of exposure to infected cattle with cattle destined for California dairies, contact with infected wildlife, exposure to humans with bTB infections, community and neighboring herds and others. Epidemiologic and molecular typing data confirmed the source of infection in 3 herds and probable sources of infection in 2 herds. In the 7 remaining herds described in this case series an epidemiologic link to a source could not be determined and molecular typing results did not associate M. bovis isolates acquired from these herds with another specific U.S. herd or U.S.-born animal. Preventing new introductions of M. bovis onto California dairies will require rigorous epidemiologic investigation of all the potential pathways of introduction discussed here. The root cause(s) of bTB on California dairies is certainly multifactorial with complex interactions of herd management practices, importation of cattle at greater risk of exposure to infected cattle, and the potential of human M. bovis exposure. The extensive use of molecular typing has improved epidemiologists' ability to narrow the scope of potential sources.

There is an intramolecular interaction in the lipid kinase Fab1 in which the upstream CCR domain contacts the Fab1 kinase region. Selected dominant-active alleles disrupt this interaction and result in elevated PI(3,5)P2. These findings suggest a regulatory mechanism that contributes to dynamic control of cellular PI(3,5)P2 synthesis., Phosphorylated phosphoinositide lipids (PPIs) are low-abundance signaling molecules that control signal transduction pathways and are necessary for cellular homeostasis. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is essential in multiple organ systems. PI(3,5)P2 is generated from PI3P by the conserved lipid kinase Fab1/PIKfyve. Defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here we report an intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We identify mutations in the kinase domain that lead to elevated levels of PI(3,5)P2 and impair the interaction between the kinase and CCR domain. We also identify mutations in the CCR domain that lead to elevated levels of PI(3,5)P2. Together these findings reveal a regulatory mechanism that involves the CCR domain of Fab1 and contributes to dynamic control of cellular PI(3,5)P2 synthesis.

SummaryAssembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.

Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs), and occurs via ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here we present the cryo-electron microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae. The positions of bound AFs were defined using cryo-EM reconstructions of pre-ribosomal complexes lacking individual components. All seven AFs are positioned to prevent each step in the translation initiation pathway by obstructing the binding sites for initiation factors, by preventing the opening of the mRNA channel, by blocking 60S subunit joining, and by disrupting the decoding site. We suggest that these highly redundant mechanisms ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation. Implications for the regulation of 40S maturation are also discussed.

Die radiolunare Arthrodese ist ein haufiger Eingriff bei Rheumapatienten. Sie wird vor allem bei schmerzhaften Sekundararthrosen im Radiokarpalbereich sowie bei palmarer Subluxation, dem Ulnarshift und der rheumatypischen Handskoliose zur Stabilisierung des Handgelenks durchgefuhrt. Mehrere Osteosyntheseverfahren wurden beschrieben. In unserem Haus wird seit Mitte 2000 die gekreuzte Schraubenosteosynthese verwendet. In unserer Nachuntersuchung sollte insbesonders uberpruft werden, ob die Komplikationsrate (v. a. Pseudarthrose und Metalldislokation) bei der gekreuzten Schraubenosteosynthese niedriger lag als bei anderen Verfahren. Daruber hinaus haben wir den Langzeitverlauf aller verwendeten Osteosyntheseverfahren verglichen. Von 1987–2005 wurden bei 57 Patienten 75 Handgelenke mittels einer radiolunaren Arthrodese teilversteift, davon 27 Handgelenke mit der gekreuzten Schraubenosteosynthese. Zur Evaluierung der Funktion von 47 Handgelenken wurde ein Fragebogen eingesetzt, 42 Handgelenke konnten klinisch nachuntersucht werden. Der mittlere Nachbeobachtungszeitraum lag bei 10,5 Jahren, mindestens jedoch bei 3 Jahren. Die Zufriedenheit der Patienten insgesamt mit dem operativen Ergebnis war hoch, in 44 von 47 Fragebogen wurde die Frage danach mit sehr zufrieden und zufrieden beantwortet. Der mittlere Disabilities of the Arm, Shoulder and Hand (DASH) Score betrug 41 Punkte. Die durchschnittliche Beweglichkeit lag bei 59° fur die Extension/Flexion. Bei der gekreuzten Schraubenosteosynthese kam es bei einem von 27 operierten Handgelenken zu einer Pseudarthrose, bei den anderen Osteosyntheseverfahren lag die Pseudarthroserate hoher. Auch Metallentfernungen wegen Dislokation mussten bei den anderen Verfahren haufiger durchgefuhrt werden. Die radiolunare Arthrodese ist eine geeignete Methode zur Stabilisierung des rheumatischen Handgelenkes. Bei der gekreuzten Schraubenosteosynthese traten deutlich weniger Komplikationen auf als bei anderen Osteosyntheseverfahren.

Purpose The advantage of wrist arthroplasty remains controversial, primarily due to the high complication rate. For this reason it seems sensible to monitor the results of different types of prostheses even with small numbers of cases. We were particularly interested to see if wrist joint arthroplasty is a useful alternative for patients with rheumatoid arthritis, and which of the types we used shows the best results. Patients and methods In our hospital, 41 wrist joint prostheses (15 Meuli, 16 BIAX and 10 Universal 2) were implanted in 36 patients from 1992 until 2005 (follow-up 1 to 14 years, mean 5.3 years). 33 patients had rheumatic destruction of the wrist, two had osteoarthritis following fracture of the scaphoid, and one pseudarthrosis after failed arthroplasty and arthrodesis for Kienbock's disease. Mean age was 54 years, ranging from 34 to 73 years. 14 patients had had surgery on this wrist before. The patients were sent a questionnaire including the DASH score, and a clinical evaluation and X-rays were performed. Results 33 patients with 38 wrist arthroplasties answered the questionnaire, 34 wrist joint prosthesis of 29 patients could be evaluated. Discussion 6 prostheses had to be removed because of complications (3 arthrodeses were performed after removal, 3 prostheses were exchanged). There were 4 dislocations (3 times with the Meuli type, once with the BIAX type). There was one case of CRPS type I. But subjectively, in answering our questionnaire, 31 of 38 patients claimed to be very satisfied or satisfied with the result of the operation, only 6 were less satisfied or not satisfied at all. An improvement of pain was found by all but one patient. An increase in strength or range of movement was found more rarely. The mean postoperative DASH score was 61 points. Mean wrist joint mobility was 50 degrees for extension/flexion, and 20 degrees for radial- and ulnar abduction. Conclusion The result of total wrist joint arthroplasty depends very much on a careful patient selection. A preoperative bony malposition of the wrist and a tendon dysfunction seem to be responsible for a bad result. High expectations with regard to range of movement and strength should be avoided. A good reduction of pain can be achieved but the risk of complication is still higher than in arthrodesis.

Background We have shown that serine proteases in German cockroach extract increase TNF-α–induced expression of IL-8 in human bronchial epithelial cells. The mechanism by which cockroach proteases regulate cytokine expression is unknown; however, protease-activated receptors (PARs) might play a role. Objective We sought to determine the role of PARs and extracellular-signal-regulated kinase (ERK) in cockroach-induced regulation of IL-8 expression. Methods 16HBE14o- human bronchial epithelial cells were treated with the specific PAR-1 and PAR-2 agonists, TFRIFD and SLIGKV, respectively. IL-8 transcription was assessed by transiently transfecting cells with a luciferase-tagged IL-8 promoter construct, and in some cases, dominant-negative expression vectors. To block PAR cleavage, antibodies against the cleavage region of PAR-1 and PAR-2 were used. ERK phosphorylation was determined by Western blot. Results Although both PAR-1 and PAR-2 were endogenously expressed in 16HBE14o- cells, selective activation of PAR-2 but not PAR-1 mimicked the effect of cockroach extract on IL-8 expression. Using a blocking antibody against cleavage of PAR-2 but not PAR-1 attenuated cockroach-extract-induced responses, suggesting that cockroach proteases cleave PAR-2. Treatment of cells with cockroach extract and SLIGKV each increased phosphorylation of ERK. Chemical or genetic inhibition of Ras and mitogen-activated protein kinase/ERK (MEK), upstream activators of ERK, each attenuated cockroach- and PAR-2–induced IL-8 transcription. Conclusion Cockroach proteases and PAR-2 activation synergistically increase TNF-α–induced IL-8 transcription via activation of ERK. These data suggest an important role for PAR-2 and ERK activation in the regulation of cytokine expression in airway epithelium in response to cockroach proteases.

Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNA⋅tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.

Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

A soy-protein isolate diet that was deficient in methionine (Met), zinc (Zn), riboflavin, vitamin B-6 and choline for chick growth (Assay 1) was used to study individual or multiple deficiencies of several of these nutrients. In all cases, adding all three deficient nutrients together resulted in growth responses that were superior to those resulting from supplementation with any pairs of deficient nutrients. In Assay 2, single addition of Zn but not of methionine or riboflavin produced a growth response, but the combination of either Zn and Met or Zn and riboflavin resulted in growth responses that were greater than the response elicited by Zn alone. Assay 3 involved individual or multiple deficiencies of choline, riboflavin and vitamin B-6, and individual additions suggested that choline was first limiting. Choline + riboflavin supplementation, however, produced marked growth and gain:food responses that were far greater than those resulting from supplemental choline or riboflavin alone. Moreover, the growth response to a combination of choline + pyridoxine (PN) was also greater than that obtained from any of the three nutrients fed alone; even PN + riboflavin (in the absence of choline) produced responses greater than those observed with the unsupplemented negative-control diet. In Assay 4, chicks responded to individual additions of riboflavin, PN or Met, and in Assay 5, to either riboflavin or PN; all two-way combinations resulted in growth rates that were far greater than those occurring with any single addition. The data from these experiments show that unlike the situation with three deficient amino acids, the expected responses to first-, second- and third-limiting B-vitamins or deficient vitamins combined with deficient levels of Zn or Met do not follow the expected pattern of response to first-, further response to first- and second- and an even further response to first-, second- and third-limiting nutrients.

Background Multidetector computed tomography (MDCT) is emerging as a powerful noninvasive diagnostic tool. The appropriate role of this technique in the preoperative evaluation of cardiovascular disease has yet to be fully defined. Atrial fibrillation is the most common sustained cardiac arrhythmia, and novel minimally invasive surgical techniques have been developed to treat this condition by electrically isolating the pulmonary veins. The ideal methodology to preoperatively evaluate these patients remains debatable. We hypothesized that 64-slice CT could significantly affect perioperative planning. Methods Thirty-six consecutive patients who consented to undergo minimally invasive pulmonary vein isolation at our institution underwent a preoperative 64-slice cardiac CT scan. All cardiac and noncardiac abnormalities were recorded, and modifications to the initial surgical plan were documented. Results The mean patient age was 64.4 ± 11.9 years [26 men (72.2%), 17 with known coronary artery disease (47.2%)]. Preoperative CT scanning detected 12 patients with abnormal pulmonary venous anatomy (33.3%), 3 with left atrial thrombus (8.3%), and 17 with significant coronary artery disease (47.2%). Furthermore, 20 studies (55.6%) detected pulmonary abnormalities (including 11 nodules). Preoperative scanning significantly altered surgical planning in 10 cases (27.8%). Alterations in patient treatment included preoperative invasive angiography, conversion of the mini-maze to an open chest procedure, alteration of surgical approach, and postponement/cancellation. Conclusions Sixty-four-slice CT scanning is a safe, rapid, and accurate procedure with important ramifications for surgical planning. This methodology could become an alternative approach to screen preoperative cardiac surgical patients.

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ϵ-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ϵ-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ϵ-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly.

The advantage of wrist arthroplasty remains controversial, primarily due to the high complication rate. For this reason it seems sensible to monitor the results of different types of prostheses even with small numbers of cases. We were particularly interested to see if wrist joint arthroplasty is a useful alternative for patients with rheumatoid arthritis, and which of the types we used shows the best results.In our hospital, 41 wrist joint prostheses (15 Meuli, 16 BIAX and 10 Universal 2) were implanted in 36 patients from 1992 until 2005 (follow-up 1 to 14 years, mean 5.3 years). 33 patients had rheumatic destruction of the wrist, two had osteoarthritis following fracture of the scaphoid, and one pseudarthrosis after failed arthroplasty and arthrodesis for Kienböck's disease. Mean age was 54 years, ranging from 34 to 73 years. 14 patients had had surgery on this wrist before. The patients were sent a questionnaire including the DASH score, and a clinical evaluation and X-rays were performed.33 patients with 38 wrist arthroplasties answered the questionnaire, 34 wrist joint prosthesis of 29 patients could be evaluated.6 prostheses had to be removed because of complications (3 arthrodeses were performed after removal, 3 prostheses were exchanged). There were 4 dislocations (3 times with the Meuli type, once with the BIAX type). There was one case of CRPS type I. But subjectively, in answering our questionnaire, 31 of 38 patients claimed to be very satisfied or satisfied with the result of the operation, only 6 were less satisfied or not satisfied at all. An improvement of pain was found by all but one patient. An increase in strength or range of movement was found more rarely. The mean postoperative DASH score was 61 points. Mean wrist joint mobility was 50 degrees for extension/flexion, and 20 degrees for radial- and ulnar abduction.The result of total wrist joint arthroplasty depends very much on a careful patient selection. A preoperative bony malposition of the wrist and a tendon dysfunction seem to be responsible for a bad result. High expectations with regard to range of movement and strength should be avoided. A good reduction of pain can be achieved but the risk of complication is still higher than in arthrodesis.

Carpal tunnel release can be performed under general or local anaesthetic. However, many surgeons believe the upper arm tourniquet is not tolerated by the patient when awake. We use a forearm tourniquet for carpal tunnel decompression under local anaesthesia. The aim of this study is to assess patient tolerance of the technique. Between January 1st 1996 and December 31st 2000, 74 patients had carpal tunnel release performed using local anaesthesia. We sent a postal questionnaire to each, asking the patient to rate different aspects of the procedure. Fifty-eight patients replied (78% response). Forty-four of the respondents (76%) tolerated the tourniquet well, finding it to be 'no problem' or only 'mildly painful'. The same number reported they would prefer to have local anaesthesia again in the event of their requiring a similar operation on their hand. We believe carpal tunnel release using local anaesthetic and a forearm tourniquet is well tolerated by the patient.

The performance of a heterodyne acoustooptical channelizer in a Mach-Zehnder configuration with an average single-channel signal-to-noise ratio (per megahertz) of 76 dB is described. The spurious signals generated by overlapping reference tones were negligible over 76 dB by virtue of the optical system design and a dual-detector cancellation scheme using an intermediate frequency of 70 MHz. Performance is described in terms of signal to noise, wideband signal uniformity, channel profile, channel isolation, and phase stability. It is concluded that heterodyne acoustooptical technologies have potential for high-performance channelizers. >

Early developmental patterning of the Drosophila embryo is driven by the activities of a diverse set of maternally and zygotically derived transcription factors, including repressors encoded by gap genes such as Krüppel, knirps, giant and the mesoderm-specific snail. The mechanism of repression by gap transcription factors is not well understood at a molecular level. Initial characterization of these transcription factors suggests that they act as short-range repressors, interfering with the activity of enhancer or promoter elements 50 to 100 bp away. To better understand the molecular mechanism of short-range repression, we have investigated the properties of the Giant gap protein. We tested the ability of endogenous Giant to repress when bound close to the transcriptional initiation site and found that Giant effectively represses a heterologous promoter when binding sites are located at −55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites are moved to more distal locations, repression is diminished. Rather than exhibiting a sharp ‘step-function’ drop-off in activity, however, repression is progressively restricted to areas of highest Giant concentration. Less than a two-fold difference in Giant protein concentration is sufficient to determine a change in transcriptional status of a target gene. This effect demonstrates that Giant protein gradients can be differentially interpreted by target promoters, depending on the exact location of the Giant binding sites within the gene. Thus, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradient. We also demonstrate that a chimeric Gal4-Giant protein lacking the basic/zipper domain can specifically repress reporter genes, suggesting that the Giant effector domain is an autonomous repression domain.

1. Histamine induces relaxation of human cranial arteries. Studies have revealed that the relaxant histamine H1-receptor predominates in human cerebral and the H2-receptor in temporal arteries, while H1- and H2-receptors are of equal importance in the middle meningeal artery. The purpose of the present study was to examine the role of the endothelium and nitric oxide in histamine-induced responses and to show the presence of mRNA encoding H1- and H2-receptors in human cranial arteries. 2. Electrophoresis of polymerase chain reaction (PCR) products from human cerebral, middle meningeal and temporal arteries, demonstrated products corresponding to mRNA encoding both H1- and H2-receptors in arteries with and without endothelium. The amplified PCR products were sequenced and showed 100% homology with the published sequences of these histamine receptors. 3. A sensitive in vitro system was used to study vasomotor responses to histamine. In precontracted cerebral, middle meningeal and temporal arteries with and without endothelium, histamine caused a concentration-dependent relaxation with Imax values between 87% and 81% and pIC50 values between 8.14 and 7.15. In arteries without endothelium the histamine-induced relaxation was significantly less potent (Imax values between 87% and 66% and pIC50 values between 7.01 and 6.67) than in cranial arteries with an intact endothelium. 4. This addition of histamine to arteries without endothelium and pretreated with the histamine H2-antagonist, cimetidine (10(-5) M), caused a concentration-dependent contraction of the cranial arteries with Emax values between 86% and 29% and pEC50 values between 7.53 and 6.77. This contraction was blocked by the histamine H1-receptor antagonist, mepyramine (10(-7) M), and even turned into a relaxation with Imax values between 84% and 14% and pIC50 values between 7.42 and 5.86. 5. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5) M) significantly inhibited the relaxant response to histamine in cerebral and temporal arteries (pIC50 values between 7.43 and 7.13). The combined treatment with L-NAME (3 x 10(-5) M) and cimetidine (10(-5) M) caused a further displacement of the concentration-response curve (pIC50 values between 7.14 and 6.57) and decreased the maximum relaxant responses in all three cranial arteries (Imax values between 62% and 39%). 6. In conclusion, this is the first study which show mRNA encoding histamine H1- and H2-receptors in human cranial arteries. The results indicate that histamine-induced relaxation of human cranial arteries is partially mediated via an endothelial H1-receptor coupled to the production of nitric oxide and partially via a H2-receptor associated with the smooth muscle cells. In addition, there is evidence for a contractile H1-receptor in the smooth muscle cells in these arteries.

In this study, we analyzed the regeneration kinetics and the reversibility of the 2.3-DPG reduction in stored red cells after transfusion. Within 3 h after transfusion the 2.3-DPG levels raised up to 40% of the patients' prior 2.3-DPG concentration, although the 2.3-DPG content of the transfused red cells was less than 10% of before. The complete regeneration of the 2.3-DPG concentration occurred after 36 to 48 h after transfusion. There was a close correlation of the results obtained by P-31-NMR and enzymatic determination of DPG.

A 60-year-old man underwent end-to-end aortobifemoral (ABF) bypass for occlusive disease. 1,2 He subsequently underwent a right-to-left femorofemoral bypass (FFB) for occlusion of the left limb of the ABF. He was referred 4 years later with fever, malaise, and purulent drainage from the right groin. A computed tomographic scan demonstrated fluid surrounding the FFB and the right limb of the ABF (A). A segment of infrarenal aorta proximal to the ABF graft appeared normal. Standard inframesocolic exposure was obtained via the previous midline incision. No fluid was evident at the graft bifurcation, and it appeared well incorporated. The residual infrarenal aorta was dissected free and prepared for an anastomosis. Openings were made in the ventromedial aspect of the obturator membrane bilaterally while standard medial exposure of the above-knee popliteal arteries was performed. Expanded polytetrafluoroethylene grafts ((W.L. Gore & Association, Inc., Tempe, Ariz) Intering; 8 mm 40 cm) were tunneled from the popliteal sites through the obturator foramina into the abdomen. (B) The limbs of a bifurcated expanded polytetrafluoroethylene graft (Gore-Tex; 18 9 mm) were cut 1 cm from the graft bifurcation, and graft-to-graft anastomoses were performed bilaterally. After systemic anticoagulation, the infrarenal aorta and the limbs of the ABF were clamped. The limbs were then transected, ligated, and advanced antegrade along the iliac tunnels. The residual body of the ABF graft was excised. The new bifurcated graft was sewn end to end to the infrarenal aorta proximally. The graft-to-graft anastomoses were assessed for hemostasis before the grafts were pulled through the previously fashioned obturator tunnels. End-to-side anastomoses were performed distally. The wounds were closed and covered with occlusive dressings before complete excision of the residual ABF limbs and the FFB via bilateral femoral and suprapubic incisions. The femoral and suprapubic incisions were packed, and the patient was maintained on chronic oral antibiotic therapy. At 24 months, he had no evidence of recurrent infection and was ambulatory at home. A follow-up computed tomographic scan demonstrated no fluid collections and patent grafts (C; cover image). The treatment of infected ABF grafts is a difficult problem. Options include in situ reconstruction with femoral vein, cryopreserved allografts, or antibiotic-impregnated synthetic grafts. Extra-anatomic reconstruction to the popliteal level is not an ideal solution secondary to poor patency. We suggest that reconstruction with synthetic bilateral obturator bypass is a viable option if there is adequate residual infrarenal aorta. This procedure allows for complete treatment of the femoral pathology and should have improved patency compared with longer extra-anatomic bypasses.

From 1982 to 1986, more than 2500 gravity data were taken in an Andean Geotraverse covering northern Chile and northwestern Argentina between 64° and 71°W and 20° and 26°S. Including 2100 reprocessed older data, there is now a data base of about 4600 gravity values available, which can be used together with other geophysical and geologicalin formation for an interdisciplinary interpretation to solve some problems of the structure and development of the Central Andes. The new gravimetric data base, which includes the Bouguer-, free-air- and isostatic residual anomaly, is presented together with a preliminary interpretation.

Crude cockroach extract elicited positive skin tests in 50% of patients with positive and in 4% with negative environmental history for cockroach exposure, suggesting a possible role of cockroach in perennial atopic disease. Three major allergens in crude American and German cockroach extracts have been identified using sequential purification steps on Sephadex G-75, diethylaminoethyl (DEAE) cellulose, and agarose gel electrophoresis. Cr-I elicits positive skin tests in 70% of patients sensitive to the crude extracts. It has a molecular weight of approximately 25,500 daltons, is highly acidic, and resists boiling for four hours. Boiling in 4 N acetic acid completely abolishes its allergenicity. The purified allergen elicits positive skin tests at a concentration of 3 μg/ml and is capable of inducing >50% histamine release from sensitive leukocytes at 0.05 ng/ml. A second antigen, Cr-II, elicits positive skin tests also in approximately 70% of cockroach-sensitive individuals, has a molecular weight of approximately 63,000 to 65,000 daltons, and has similar heat stability and acid hydrolysis characteristics to Cr-I. A third, less well-characterized antigen, Cr-III, has a molecular weight

The giant protein is a short-range transcriptional repressor that refines the expression pattern of gap and pair-rule genes in the Drosophila blastoderm embryo. Short-range repressors including knirps, Kruppel, and snail utilize the CtBP cofactor for repression, but it is not known whether a functional interaction with CtBP is a general property of all short-range repressors. We studied giant repression activity in a CtBP mutant and find that this cofactor is required for giant repression of some, but not all, genes. While targets of giant such as the even-skipped stripe 2 enhancer and a synthetic lacZ reporter show clear derepression in the CtBP mutant, another giant target, the hunchback gene, is expressed normally. A more complex situation is seen with regulation of the Kruppel gene, in which one enhancer is repressed by giant in a CtBP-dependent manner, while another is repressed in a CtBP-independent manner. These results demonstrate that giant can repress both via CtBP-dependent and CtBP-independent pathways, and that promoter context is critical for determining giant-CtBP functional interaction. To initiate mechanistic studies of the giant repression activity, we have identified a minimal repression domain within giant that encompasses residues 89–205, including an evolutionarily conserved region bearing a putative CtBP binding motif.

Classical enzyme kinetic analyses were applied to define the mechanism of the effect of heparin on the cleavage of the fourth (C4) and second (C2) components of complement by the first component (C1) of complement. The data indicated that heparin was a noncompetitive inhibitor of both C4 and C2 cleavage; cleavage of C2 was more sensitive to the action of heparin than cleavage of C4. Heparin blocked the action of C 1 in serum and three preparations of purified C 1 , each isolated by different methods. Heparin also inhibited the increased vascular permeability produced in normal skin by activated C 1 , suggesting that it or other polyions might be effective in the treatment of hereditary angioneurotic edema attacks.

Crude cockroach extract elicited positive skin tests in 50% of patients with positive and in 4% with negative environmental history for cockroach exposure, suggesting a possible role of cockroach in perennial atopic disease. Three major allergens in crude American and German cockroach extracts have been identified using sequential purification steps on Sephadex G-75, diethylaminoethyl (DEAE) cellulose, and agarose gel electrophoresis. Cr-I elicits positive skin tests in 70% of patients sensitive to the crude extracts. It has a molecular weight of approximately 25,500 daltons, is highly acidic, and resists boiling for four hours. Boiling in 4 N acetic acid completely abolishes its allergenicity. The purified allergen elicits positive skin tests at a concentration of 3 mug/ml and is capable of inducing greater than 50% histamine release from sensitive leukocytes at 0.05 ng/ml. A second antigen, Cr-II, elicits positive skin tests also in approximately 70% of cockroach-sensitive individuals, has a molecular weight of approximately 63,000 to 65,000 daltons, and has similar heat stability and acid hydrolysis characteristics to Cr-I. A third, less well-characterized antigen, Cr-III, has a molecular weight less than 10,000 daltons and elicits positive skin tests in 30% of individuals sensitive to the crude extract.

Virus-like structures were present in all of 42 renal biopsies from 35 patients with SLE, five of whom had normal kidney tissue by light microscopy. Structures were also present in biopsies from 24 of 113 patients with renal disease other than SLE who had been selected at random. Fourteen of these were children, and three had normal kidney tissue by light microscopy. In biopsies from eight normal volunteers, there was no evidence of the structures. Although the structures were uniformly present in the kidneys of patients with SLE, they were not specific for SLE.

Background: Posttransfusional changes of preserved red blood cells can influence the oxygen equilibrium curve which is mainly affected by the concentration of erythrocyte 2,3-diphosphoglycerate (DPG). Material and Methods: The regeneration kinetics of DPG and nucleotides (ATP, ADP, AMP, GTP, GDP) was determined over a period of 0-48 h in surgically treated patients following transfusion of DPG-depleted packed red cells stored for 14 days in CPD-SAGM. Results: 3 h after transfusion the DPG levels raised up to 40% of the patients’ prior DPG concentrations. Complete regeneration of the DPG concentrations occurred 36-48 h after transfusion. Changes in the nucleotide pattern indicate, after a temporary decrease of ATP and GTP levels (after 10-30 min) and an activation phase (after 3-12 h), the full regeneration of these parameters 24-48 h after transfusion. Conclusions: The regeneration kinetics of DPG should be taken into consideration for transfusions with blood units stored for more than 14 days, especially in patients with reduced compensatory mechanisms (coronary and cerebral scleroses, pacemaker, etc.) and large transfusion volumes.

Lysosomes provide a major source for cellular cholesterol; however, most of this cholesterol is trafficked to the plasma membrane via unknown mechanisms. In this issue of Cell, Chu et al. identify an unexpected role for peroxisomes in the transport of cholesterol from the lysosome to the plasma membrane via a lysosome-peroxisome membrane contact site.

SET domain lysine methyltransferases (KMTs) are S-adenosylmethionine (AdoMet)-dependent enzymes that catalyze the site-specific methylation of lysine residues in histones, transcription factors, chromatin modifying enzymes, and other protein substrates. These modifications mediate protein:protein interactions with signaling factors that possess effector modules which can recognize methyllysines in a sequence-dependent manner. In addition to their site specificity, SET domain KMTs exhibit product specificity, which is defined as their ability to catalyze different degrees of methylation of the lysine epsilon amine group, thus imparting an additional hierarchy in methyllysine signaling. To understand the mechanism underlying the product specificity of SET domain KMTs, we have structurally and functionally characterized two active site mutants of the human monomethylase SET7/9 that alter its specificity to a dimethylase and a trimethylase, respectively. Structures of the SET7/9 mutants in complex with peptides bearing unmodified, mono, di, and trimethylated lysines reveal that water molecules within the active site function as molecular place holders that align the lysine epsilon amine group in a linear geometry with the sulfonium methyl group of AdoMet to promote methyltransfer. As the methylation state of the lysine substrate increases during successive methyltransfer reactions, the water molecules dissociate from the active site, thereby enlarging the lysine binding channel to accommodate the increasing bulk of the multiply methylated epsilon amine group. Taken together, our findings illuminate the catalytic roles of active site water molecules in facilitating lysine multiple methylation by SET domain KMTs.