A comparison of port-site infiltration with bupivacaine alone vs. bupivacaine plus dexemedetomidine after diagnostic staging laparoscopy.

Objectives: To compare the effects of bupivacaine alone and in combination with dexmedetomidine following staging laparoscopies. METHODS: This triple-blinded, prospective study was conducted from June to September 2021 at a tertiary care cancer hospital in Lahore, Pakistan, and comprised adult patients having American Society of Anaesthesiologists grade I-III, weighing >30kg and undergoing diagnostic staging laparoscopy. The subjects were randomised into two equal groups. Group A received 6ml of 2mg/kg bupivacaine at each of the four laparoscopic port sites before skin closure, while group B additionally received 2µg/kg dexmedetomidine. The presence and severity of pain were recorded and assessed at 15 min, 1, 2 and 4 hours as well as at the time of discharge from the post-anaesthesia care unit. The time to first request for rescue analgesia, total morphine consumption, and the occurrence of any side effects during their stay were also recorded. Data was analysed using SPSS 23. RESULTS: Of the 30 patients, 15(50%) were in group A; 10(66.6%) males and 5(33.3%) females with mean age 43.27±7.59 years. There were 15(50%) patients in group B; 12(80%) males and 3(20%) females with mean age 41.36±12.42 years (p>0.05). Of the total, 29(96.66%) patients were classified as American Society of Anaesthesiologists grade II, and 1(3.33%) patient in group A was grade III. There was no significant difference between the groups in any of the outcome measures assessed (p>0.05), and none of the patients experienced any side effect throughout the post-operative stay. CONCLUSIONS: The combination of dexmedetomidine and bupivacaine had no significant improvement in pain relief compared to bupivacaine alone.

Identification of the Viral Determinant of Hypovirulence and Host Range in Sclerotiniaceae of a Genomovirus Reconstructed from the Plant Metagenome.

Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified in the newly formed family Genomoviridae. Here, we determined the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fructicola, two economically important members of the family Sclerotiniaceae, and Fusarium oxysporum. SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea, and M. fructicola. SlaGemV-1 also replicates in Spodoptera frugiperda insect cells but not in Caenorhabditis elegans or plants. By expressing viral genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. IMPORTANCE Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant-pathogenic fungi and fall armyworm (S. frugiperda Sf9) insect cells but not a model nematode, C. elegans, or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species in the family Sclerotiniaceae, including Sclerotinia sclerotiorum, Botrytis cinerea, and Monilinia fructicola. The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenomes can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.

Patient with ataxia telangiectasia undergoing elective staging laparoscopy: A case report and literature review.

Ataxia telangiectasia is a rare autosomal recessive condition which develops due to a mutation in the ataxia telangiectasia mutated gene (ATM gene). As a result of this mutation, the ability of the DNA to undergo repair is undermined. The resulting cellular demise is responsible for the diverse presentation of the clinical condition. Neurological symptoms such as cerebellar ataxia, abnormal eye movements and malignancies occur commonly, while immunodeficiency predisposes these patients to recurrent infections. Perioperative management of patients with this rare condition can be associated with increased morbidity. Therefore, it is recommended that patients with ataxia telangiectasia should be managed in a multidisciplinary centre, under the supervision of senior clinicians who have the insight into the clinical needs of such patients. We report herein, the perioperative management of a patient with ataxia telangiectasia undergoing laparoscopic procedure. This case report will allow the readers to increase their knowledge and understanding when it comes to the management of these patients.

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus.

To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region in vitro by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA invivo Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation.IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.

Investigation of salt tolerance in cotton germplasm by analyzing agro-physiological traits and ERF genes expression.

The development of genotypes that can tolerate high levels of salt is crucial for the efficient use of salt-affected land and for enhancing crop productivity worldwide. Therefore, incorporating salinity tolerance is a critical trait that crops must possess. Salt resistance is a complex character, controlled by multiple genes both physiologically and genetically. To examine the genetic foundation of salt tolerance, we assessed 16 F1 hybrids and their eight parental lines under normal and salt stress (15 dS/m) conditions. Under salt stress conditions significant reduction was observed for plant height (PH), bolls/plant (NBP), boll weight (BW), seed cotton yield (SCY), lint% (LP), fiber length (FL), fiber strength (FS), potassium to sodium ratio (K+/Na+), potassium contents (K+), total soluble proteins (TSP), carotenoids (Car) and chlorophyll contents. Furthermore, the mean values for hydrogen peroxide (H2O2), sodium contents (Na+), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and fiber fineness (FF) were increased under salt stress. Moderate to high heritability and genetic advancement was observed for NBP, BW, LP, SCY, K+/Na+, SOD, CAT, POD, Car, TSP, FL, and FS. Mean performance and multivariate analysis of 24 cotton genotypes based on various agro-physiological and biochemical parameters suggested that the genotypes FBS-Falcon, Barani-333, JSQ-White Hold, Ghauri, along with crosses FBS-FALCON × JSQ-White Hold, FBG-222 × FBG-333, FBG-222 × Barani-222, and Barani-333 × FBG-333 achieved the maximum values for K+/Na+, K+, TSP, POD, Chlb, CAT, Car, LP, FS, FL, PH, NBP, BW, and SCY under salt stress and declared as salt resistant genotypes. The above-mentioned genotypes also showed relatively higher expression levels of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 at 15 dS/m and proved the role of these ERF genes in salt tolerance in cotton. These findings suggest that these genotypes have the potential for the development of salt-tolerant cotton varieties with desirable fiber quality traits.

Drought and heat stress on cotton genotypes suggested agro-physiological and biochemical features for climate resilience.

This study aimed to investigate the impact of individual drought, heat, and combined drought and heat stress on twelve cotton genotypes, including eight tolerant and four susceptible genotypes. A field experiment was carried out by employing a randomized complete block split-plot design, with treatments (control, drought, heat, drought + heat), and cotton genotypes assigned to the main plots and sub-plots respectively. The results showed that the combined stress had a more severe impact on the yield and fiber quality of cotton genotypes compared to individual stresses. Among the studied genotypes, FB-Shaheen, FH-207, MNH-886, and White Gold exhibited superior performance in regard to agronomic and fiber quality characters under combined stress environments. Physiological parameters, including transpiration rate, stomatal conductance, relative water contents, and photosynthetic rate, were significantly reduced under combined stress. However, specific genotypes, MNH-886, FH-207, White Gold, and FB-Shaheen, demonstrated better maintenance of these parameters, indicating their enhanced tolerance to the combined stress. Furthermore, the accumulation of reactive oxygen species was more pronounced under combined stress compared to individual stressors. Tolerant genotypes showed lower levels of H2O2 and MDA accumulation, while susceptible genotypes exhibited higher levels of oxidative damage. Antioxidant enzyme activities, such as superoxide dismutase, peroxidase, and catalase, increased under combined stress, with tolerant genotypes displaying higher enzyme activities. Conversely, susceptible genotypes (AA-703, KZ 191, IR-6, and S-15) demonstrated lower increases in enzymatic activities under combined stress conditions. Biochemical traits, including proline, total phenolic content, flavonoids, and ascorbic acid, exhibited higher levels in resistant genotypes under combined stress, while sensitive genotypes displayed decreased levels of these traits. Additionally, chlorophyll a & b, and carotenoid levels were notably decreased under combined stress, with tolerant genotypes experiencing a lesser decrease compared to susceptible genotypes.

Emergency Management of Ventilation Failure Through Blocked Tracheostomy Tube in a Paediatric Patient.

The most common complication of tracheostomy tubes in children is blockage of the tube. We report a case where ventilation after induction of anaesthesia was not possible even though there were no signs of impending obstruction. An eight-year-old child, recently diagnosed with left tonsillar embryonal rhabdomyosarcoma, presented for an MRI face and bone marrow biopsy before starting treatment. Due to difficulty in breathing, the patient had undergone a tracheostomy at a different institute and a size six uncuffed tracheostomy tube was in situ. There was difficulty in ventilating the patient due to blockage in the tracheostomy tube which was addressed and the patient was discharged after successful completion of both the procedures. This case highlights the importance of following an emergency algorithm for failure to ventilate in a patient with a tracheostomy tube, identifying the cause and treating it.

Improvement in Durability and Mechanical Performance of Concrete Exposed to Aggressive Environments by Using Polymer.

Concrete is the most widely used construction material. However, it cannot sustain the harsh environment and can easily deteriorate. It results in repair and reworks that amount to a considerable loss of money and time. The life span of concrete reduces if exposed to external attacks, for instance, sulfate attacks, alkali-silica reactions, corrosion, and drying shrinkage. These ubiquitous attacks cause a reduction in service life and raise the need for early repair and maintenance, resulting in higher life cycle costs and structural failures. To resolve these issues, the potential of styrene-butadiene-rubber (SBR) ultrafine powder as cement replacement polymeric admixture at 0%, 3%, 5%, 7%, and 10% have been evaluated. The effect of SBR-powder on concrete is investigated by conducting an alkali-silica reactivity test (ASR), rapid-chloride-permeability test (RCPT), drying shrinkage, and sulfate resistivity tests. Workability, compressive and flexural strength tests are also conducted. For ASR and drying shrinkage, mortar bar samples were cast, exposed to respective environments, and the percentage change in length was measured. For mechanical tests and RCPT, prisms, cylinders and cubes were cast and tested at 28 days. The SBR-powder modification reduces concrete's permeability, drying shrinkage, and expansions due to ASR and sulfate attacks. SBR powder increased workability by 90%, compressive strength by 23%, and flexural strength by 9.4% in concrete when used at 10% cement replacement by weight. The SBR-powder (10%) modification reduced the RCPT value by up to one-third (67%), drying shrinkage by 53%, ASR by 57%, and sulfate reaction by 73%. Consequently, SBR powder usage can adequately improve the workability, mechanical properties, and durability of the concrete and lead to advanced sustainable concrete with low repair requirements.

Implementation of meta-analysis approach, comparing conventional radiotherapy, and proton beam therapy treating head and neck cancer.

INTRODUCTION: Radiation therapy is commonly used in the treatment of head and neck cancer in both the definitive and postoperative settings. Proton therapy, due to its intrinsic physical properties, has the ability to reduce the integral dose delivered to the patients while maintaining highly conformal target coverage. MATERIALS AND METHODS: .A literature search was performed on scientific databases, and Preferred Reporting Items for Meta-Analyses guidelines were followed to compute results. Only original studies were selected. Selected studies were used to extract some proposed data for comparison, dosimetry, site, complications, and survival. RESULTS: Proton beam therapy technology can be used against the conventional radiotherapy and shows satisfactory results. Yet conventional therapy is not less advantageous considering the amount of work available for any cross interpretations. CONCLUSION: Comparative preplanning could be beneficial considering multiple therapies for ruling out the best treatment outcomes that could be expected.

Surgical Attitudes And Preferences Regarding Choice Of Anaesthesia Amongst Orthopaedic Surgeons.

BACKGROUND: Regional anaesthesia is choice of anaesthesia for orthopaedic surgery due to lower side effects such as nausea, vomiting, postoperative pain and early mobilization. Despite of this, some orthopaedic patients refuse this modality. This study was conducted to interrogate the surgeons about their choice of anaesthesia in order to gain some insights into the concerns of surgeons and to change their minds and choose a safer mode of anaesthesia. The aim of the study was to assess the surgeons' fears and their perception about regional anaesthesia. METHODS: After institutional approval, thirty surgeons from three different tertiary care hospitals were interviewed. They were asked questions as per the questionnaire to choose choice of anaesthesia for their patients. RESULTS: After regional anaesthesia, 27.6% of respondents were concerned about paralysis and neurological disorders, 34.5% about seeing and hearing whatever is happening in theatre, 17.2% about perioperative pain, 24.1% about backache and 24.1% about delayed discharge. The most important reason to change their mind was the reassurance that the complications are not as frequently as they thought their patient would receive after a block. CONCLUSIONS: Due to the lack of information on regional anaesthesia and the risks of general anaesthesia, orthopaedic surgeons' fears and conceptions about regional anaesthesia are distorted. Anaesthesiologists should be aware of the concerns of the surgeons as well as the patients and should be willing to discuss the concerns with them and suggest the safest way to receive anaesthesia with evidence-based data.

General Anaesthesia Versus Regional Anaesthesia For Lumbar Laminectomy: A Review Of The Modern Literature.

BACKGROUND: Lumbar spine surgery can be performed using different anaesthetic techniques such as general endotracheal anaesthesia (GA) or spinal-based regional anaesthesia (RA). Few of the studies have been done to compare the outcomes of spinal anaesthesia versus general anaesthesia for lumbar laminectomies as both having some advantages as well as disadvantages but still it is controversial. The objective of current study is to make a comprehensive review of literature for comparing the outcomes of lumbar laminectomy performed under general anaesthesia versus spinal anaesthesia. METHODS: Literature search was performed by using PubMed, Google scholar and bibliography of related articles. To compare groups of general anaesthesia versus spinal anaesthesia, the variables focused were mean heart rate (HR), mean arterial pressure (MAP), blood loss during surgery, duration of surgery, post-operative anaesthesia care unit (PACU) time, postoperative narcotic use/pain scale, post-operative urinary retention, and post-operative nausea/vomiting. RESULTS: Data of eleven studies were presented in current article, of these five were randomized controlled trials, three case-controls and four were retrospective cohort studies. 5/8 studies reported that SA group having more hemodynamic stability with postoperative outcomes as compared to GA. Likewise, majority of reviewed studies (7/8) reported better pain control or decreased requirement of analgesics in SA group. Additionally, more than half of the reviewed studies (5/8) reported lower incidence of postoperative nausea and vomiting among patients of SA group.. CONCLUSIONS: The current study concluded that SA has better outcomes than GA in terms of hemodynamic stability and decrease postoperative adverse effects. So special attention should be paid for SA as an alternative to GA for lumbar laminectomy.

Life Threatening Anaphylaxis With Co-Amoxiclav Despite Having A Previous Tolerant Exposure To The Antibiotic.

Co-amoxiclav is used as a routine prophylactic antibiotic in surgical procedures. We present a case of 61 years old lady came to outpatient clinic with a diagnosis of right sided lower alveolar tumour. A plan was made to proceed with a Right Marginal Mandibulectomy. She received Co amoxiclav as prophylactic antibiotic and was clinically uneventful. On further questioning there were many different occasions on which the patient had oral co amoxiclav with no complications or side effects. No known drug allergies or otherwise were documented. As a part of surgical antibiotic prophylaxis, Co amoxiclav 1.2 gm was given to patient at the time of induction. After the antibiotic administration patient started having bronchospasm with increased airway pressures and severe hypotension indicating anaphylactic reaction. The patient was resuscitated, recovered from the anaphylactic shock successfully and provided extended recovery in post anaesthesia care unit followed by High Dependency Unit for overnight monitoring. Every centre or ward that administers injectable antibiotics have to have a standard protocol for the management of allergy reactions. A timely fashion is mandatory to make the best use of the drugs available and emergency gadget ought to be in vicinity in case this type of state of affairs rise up.

Roles of Argonautes and Dicers on Sclerotinia sclerotiorum Antiviral RNA Silencing.

RNA silencing or RNA interference (RNAi) is an essential mechanism in animals, fungi, and plants that functions in gene regulation and defense against foreign nucleic acids. In fungi, RNA silencing has been shown to function primarily in defense against invasive nucleic acids. We previously determined that mycoviruses are triggers and targets of RNA silencing in Sclerotinia sclerotiorum. However, recent progresses in RNAi or dsRNA-based pest control requires more detailed characterization of the RNA silencing pathways in S. sclerotiorum to investigate the utility of dsRNA-based strategy for white mold control. This study elucidates the roles of argonaute enzymes, agl-2 and agl-4, in small RNA metabolism in S. sclerotiorum. Gene disruption mutants of agl-2 and agl-4 were compared for changes in phenotype, virulence, viral susceptibility, and small RNA profiles. The Δagl-2 mutant but not the Δagl-4 mutant had significantly slower growth and virulence prior to virus infection. Similarly, the Δagl-2 mutant but not the Δagl-4 mutant, showed greater debilitation under virus infection compared to uninfected strains. The responses were confirmed in complementation studies and revealed the antiviral role of agl-2. Gene disruption mutants of agl-2, agl-4, Dicer-like (dcl)-1, and dcl-2 did not change the stability of the most abundant endogenous small RNAs, which suggests the existence of alternative enzymes/pathways for small RNA biogenesis in S. sclerotiorum. Furthermore, in vitro synthesized dsRNA targeting agl-2 showed a significantly reduced average lesion diameter (P < 0.05) on canola leaves with agl-2 down-regulated compared to controls. This is the first report describing the effectiveness of RNA pesticides targeting S. sclerotiorum RNA silencing pathway for the control of the economically important pathogen.

Effect Of Dexamethasone Versus Placebo For Improved Recovery In Patients Undergoing Anaesthetic Interventions For General Surgical Procedures.

BACKGROUND: Endotracheal intubation is one of the basic prerequisites of general anaesthesia. Recovery of patients is delayed due to the development of post-operative nausea, vomiting, sore throat and shivering. This study was conducted to determine role of dexamethasone for improved recovery of patients. METHODS: This randomized controlled trial was performed in the Department of Anaesthesia, Ayub Teaching Hospital, Abbottabad from June to November 2018. One hundred and twenty-two consenting patients of both genders, aged between 18-60 years, American Society of Anaesthesiologists (ASA) I and II, scheduled for general surgeries of 60-180 minutes duration under general anaesthesia were included. Patients with long duration surgeries, anticipated difficult airways, nasogastric tube in situ, upper respiratory tract infections, on steroid therapy and critically ill and emergency surgeries were excluded. They were randomly allocated into two groups by lottery method as dexamethasone (group A, n=61) or Placebo (group B, n=61). Postoperative nausea, vomiting, sore throat was observed at 2, 12 and 24 hours, shivering was observed at 2 hours only and patient satisfaction at 24 hours post-operatively. RESULTS: Group A patients had statistically proven better outcomes at 2, 12 and 24 hours for post-operative sore throat, nausea and vomiting, shivering was controlled in 24.5% p-value (0.006). Patient satisfaction was found in 83.6% of group A patients.. CONCLUSIONS: Thus, dexamethasone is effective drug for prevention of postoperative nausea, vomiting, sore throat and shivering in general surgical procedures thus improving patient satisfaction and their early discharge.

Massive Haemorrhage In Patient After Repair Of Post Esophagectomy Diaphragmatic Hernia And A Loading Dose Of Clopidogrel.

Postoperative diaphragmatic hernia (PDH) is an increasingly reported complication of esophageal cancer surgery. It occurs more frequently with minimally invasive techniques and very little is known about its pathogenesis. Currently, no consensus exists concerning preventive measures and its management. A 36 years old male underwent minimally invasive esophagectomy and presented with chest pain and dyspnoea in the emergency department 8 months after the procedure. He was started on acute coronary syndrome treatment protocol but was later diagnosed on CT scan to have diaphragmatic hernia through a defect in the oesophageal hiatus. As he was already loaded with dual anti platelet therapy it led to qualitative defect of the platelet which resulted in bleeding post procedure and the patient needed resuscitation with blood products. Dual anti-platelet therapy is an integral component of early management strategy in acute coronary syndrome. Chest X-ray was not helpful, but abdominal or chest computed tomography was useful for accurate diagnosis.

Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses.

The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent.

Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor.

BACKGROUND AND PURPOSE: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca(2+) channels. Interactions of the commonly used antagonists of IP3Rs with IP3R subtypes are poorly understood. EXPERIMENTAL APPROACH: IP3-evoked Ca(2+) release from permeabilized DT40 cells stably expressing single subtypes of mammalian IP3R was measured using a luminal Ca(2+) indicator. The effects of commonly used antagonists on IP3-evoked Ca(2+) release and (3) H-IP3 binding were characterized. KEY RESULTS: Functional analyses showed that heparin was a competitive antagonist of all IP3R subtypes with different affinities for each (IP3R3 > IP3R1 ≥ IP3R2). This sequence did not match the affinities for heparin binding to the isolated N-terminal from each IP3R subtype. 2-aminoethoxydiphenyl borate (2-APB) and high concentrations of caffeine selectively inhibited IP3R1 without affecting IP3 binding. Neither Xestospongin C nor Xestospongin D effectively inhibited IP3-evoked Ca(2+) release via any IP3R subtype. CONCLUSIONS AND IMPLICATIONS: Heparin competes with IP3, but its access to the IP3-binding core is substantially hindered by additional IP3R residues. These interactions may contribute to its modest selectivity for IP3R3. Practicable concentrations of caffeine and 2-APB inhibit only IP3R1. Xestospongins do not appear to be effective antagonists of IP3Rs.

Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by adenophostin A and its analogues.

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca(2+) channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and ß-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca(2+) release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

Stimulation of inositol 1,4,5-trisphosphate (IP3) receptor subtypes by analogues of IP3.

Most animal cells express mixtures of the three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R) encoded by vertebrate genomes. Activation of each subtype by different agonists has not hitherto been examined in cells expressing defined homogenous populations of IP(3)R. Here we measure Ca(2+) release evoked by synthetic analogues of IP(3) using a Ca(2+) indicator within the lumen of the endoplasmic reticulum of permeabilized DT40 cells stably expressing single subtypes of mammalian IP(3)R. Phosphorylation of (1,4,5)IP(3) to (1,3,4,5)IP(4) reduced potency by ~100-fold. Relative to (1,4,5)IP(3), the potencies of IP(3) analogues modified at the 1-position (malachite green (1,4,5)IP(3)), 2-position (2-deoxy(1,4,5)IP(3)) or 3-position (3-deoxy(1,4,5)IP(3), (1,3,4,5)IP(4)) were similar for each IP(3)R subtype. The potency of an analogue, (1,4,6)IP(3), in which the orientations of the 2- and 3-hydroxyl groups were inverted, was also reduced similarly for all three IP(3)R subtypes. Most analogues of IP(3) interact similarly with the three IP(3)R subtypes, but the decrease in potency accompanying removal of the 1-phosphate from (1,4,5)IP(3) was least for IP(3)R3. Addition of a large chromophore (malachite green) to the 1-phosphate of (1,4,5)IP(3) only modestly reduced potency suggesting that similar analogues could be used to measure (1,4,5)IP(3) binding optically. These data provide the first structure-activity analyses of key IP(3) analogues using homogenous populations of each mammalian IP(3)R subtype. They demonstrate broadly similar structure-activity relationships for all mammalian IP(3)R subtypes and establish the potential utility of (1,4,5)IP(3) analogues with chromophores attached to the 1-position.