Additivity of molecular fields: CoMFA study on dual activators of PPARalpha and PPARgamma

Recent trends in drug discovery include methods to identify dual and triple activating drugs. This approach is being successfully employed in malaria, cancer, asthma, insulin resistance, etc. Molecular field analysis has been employed in correlating pharmacological data and field parameters. In this paper we introduce the concept of additivity of molecular fields to correlate molecular fields of dual activators and their pIC(50) values. PPARalpha and PPARgamma dual activators, which affect hypertriglyceridemia and hyperglycemia, have been chosen to validate the molecular field additivity concept. Three CoMFA models namely alpha-model, gamma-model and dual-model have been developed. The validity of this concept has been ascertained by (a) comparing contour maps, (b) by comparing CoMFA results with FlexX docking results and (c) by analyzing newly designed molecules.     

Imbalance between Angiotensin II - Angiotensin (1-7) system is associated with vascular endothelial dysfunction and inflammation in type 2 diabetes with newly diagnosed hypertension

AimDiabetes is associated with Renin-angiotensin-aldosterone-system (RAAS) activation. Protective role of Angiotensin (1-7) has been recently identified. The study aims to identify associations between imbalance in RAAS components with vascular endothelial dysfunction and inflammation in diabetics with newly diagnosed hypertension.MethodsBrachial Flow-mediated-dilation (FMD), Carotid Intima-media-thickness (CIMT), pulse-wave-velocity (PWV), Serum E-selectin, Vascular-Cell-Adhesion-Molecule-1 (VCAM-1), high-sensitivity C-Reactive Protein (hsCRP), Interleukin-10 (IL-10), Renin, AngiotensinII, Angiotensin-Converting-Enzyme 2 (ACE2) and Angiotensin1-7 were measured in 60 diabetic patients with newly diagnosed hypertension. Patients with AngiotensinII/Angiotensin1-7 ratio <1 were classified as Favourable-Axis (FA) group (n = 22) and those with ratio >1 were classified as Unfavourable-Axis (UA) group (n = 38).ResultshsCRP was higher [9.52 (4.64–16.19) vs 3.62 (1.77–13.09) (mg/l), p = 0.04], IL-10 was lower [2.26 (1.34–12.05) vs 10.98 (4.44–17.78) (pg/ml),p = 0.006], %FMD was lower [(5.51 ± 2.97) vs (7.66 ± 3.38) (%), p = 0.01] and CIMT was higher in UA compared to FA group [0.7 (0.55–0.79) vs 0.51 (0.49–0.65) (mm), p = 0.001]. Renin correlated positively with pressure, PWV, E-selectin and VCAM-1, opposing associations were obtained for Angiotensin1-7 and ACE2.ConclusionImbalance between AngiotensinII – Angiotensin1-7 is associated with increased inflammation and vascular dysfunction in diabetics and can contribute to development of hypertension in these patients.     

Late presentation of fractures of the lateral condyle of the humerus in children

Background:

The current controversy regarding the management of fractures of the lateral condyle of the humerus presenting between 3 to 12 weeks prompted us to evaluate our results of open reduction and internal fixation of such fractures.

Patients and Methods:

Twenty-one patients operated between March 1995 and February 2001 qualified for this study. Five patients presented between 3–4 weeks, nine between 5–8 weeks and seven between 9–12 weeks post injury. Ten fractures were classified as stage II and eleven as stage III (Jacob et al. criteria). The mean age was 8 years (range: 4–14 years). All patients underwent surgery (open reduction and internal fixation with K-wires/screw, with or without bone grafting). The results were assessed by the modified criteria of Agarwal et al. after an average follow-up of 2.3 years.

Results:

Excellent to good results were observed in all the five patients presenting at 3–4 weeks post injury. In the patients presenting at 5–8 weeks, the results were excellent in one, good in four, fair in three, and poor in one patient. The fracture united in all cases; however, malunion was observed in four patients. The fractures that were operated at 9–12 weeks showed good results in one case, fair result in three cases, and poor result in three cases. Avascular necrosis of the lateral condyle in one patient, premature fusion in two patients, pin tract infection in three patients, and gross restriction of elbow movements in three patients were the major complications in this group. Accurate reduction was difficult as a result of new bone formation and remodeling at the fracture surfaces. Multiple incisions over the common extensor aponeurosis and bone graft supplementation were helpful for achieving acceptable reduction.

Conclusion:

Open reduction and internal fixation is recommended in all cases of displaced fractures of the lateral condyle of the humerus presenting at up to 12 weeks post injury. However, the results become poorer with increase in duration after injury and the grade of displacement. To avoid complications it is important to carry out careful dissection of the soft tissue attachments and to mobilize the fragment without the use of force.     

The use of ABO-incompatible grafts in living donor liver transplantation—First report from India

ABO incompatibility is the commonest reason for rejection of donors in living donor liver transplantation (LDLT). The donor pool could be expanded by 25 % to 35 % if the ABO barrier is overcome. In the absence of pre-conditioning, transplantation across the blood groups is fraught with the almost universal risk of antibody-mediated rejection (AMR) that rapidly leads to graft loss. However, AMR can be prevented by removal of preformed antibodies and reducing their production by B cells. We describe our initial experience of three cases of ABO-incompatible (ABO-i) LDLT: a 42-year-old male, an 8-month-old male and a 28-month-old female, all of blood group O+ who received blood group B + right lobe, B + left lateral segment, and A + left lateral segment liver grafts, respectively. Pre-LDLT conditioning included administration of anti-CD20 antibody (Rituximab^®) to the adult 4 weeks prior, and four to seven sessions of double-filtration plasmapheresis to all, to remove preformed antibodies and achieve anti-donor blood group antibody (ADA) titers of ≤1:16 IgG and ≤1:8 IgM, respectively. In addition, cases 1 and 3 received mycophenolate mofetil for 7 days prior to LDLT. After LDLT, all three patients achieved normal graft function over 8–17 days with no evidence of AMR and without the need for further plasmapheresis. Postoperative complications included portal vein thrombosis (one successfully re-explored), CMV (one), Pseudomonas and Klebsiella sepsis (one each), and abdominal collection (one treated with percutaneous drainage). All are currently well with normal graft function and low ADA titers at 8, 16, and 19 months after ABO-i LDLT.     

HNH proteins are a widespread component of phage DNA packaging machines

The genome packaging reactions of tailed bacteriophages and herpes viruses require the activity of a terminase enzyme, which is comprised of large and small subunits. Phage genomes are replicated as linear concatemers composed of multiple copies of the genome joined end to end. As the terminase enzyme packages the genome into the phage capsid, it cleaves the DNA into single genome-length units. In this work, we show that the phage HK97 HNH protein, gp74, is required for the specific endonuclease activity of HK97 terminase and is essential for phage head morphogenesis. HNH proteins are a very common family of proteins generally associated with nuclease activity that are found in all kingdoms of life. We show that the activity of gp74 in terminase-mediated cleavage of the phage cos site relies on the presence of an HNH motif active-site residue, and that the large subunit of HK97 terminase physically interacts with gp74. Bioinformatic analysis reveals that the role of HNH proteins in terminase function is widespread among long-tailed phages and is uniquely required for the activity of the Terminase_1 family of large terminase proteins.Tailed bacteriophages and herpes viruses package their large double-stranded DNA genomes into a preformed protein shell, known as the “prohead,” using terminase enzymes. In both types of viruses, the genome is synthesized as concatemers composed of multiple copies of the genome joined end to end. This concatemeric DNA is packaged into the prohead and cleaved into genome-length units by terminase in an ATP-dependent reaction. Phage terminases are composed of two proteins: the large subunit harbors an endonuclease domain and an ATPase that powers the DNA packaging reaction, and the small subunit mediates specific DNA-binding required for recognition of packaging sites in the phage genome. A variety of elegant structural and biophysical studies have recently provided insight into the molecular mechanisms of terminase function (1, 2). However, the factors that affect the action of terminase enzymes in vivo have been less well characterized.Terminase enzymes perform several functions. They specifically recognize and bind the viral genome, interact with the prohead, then drive the DNA into the head through the narrow entry channel formed by the portal protein that is positioned at a single vertex of the head. During this process terminases also cleave the viral DNA, either nonspecifically upon head filling or at a specific site known as “cos.” The efficient packaging of a phage genome in vivo may require phage-encoded cofactors in addition to the terminase enzyme. For example, Escherichia coli phage λ gpFI facilitates interaction of the terminase–DNA complex with proheads (3–6). A wide variety of phages appear to encode proteins with a function similar to λ gpFI (7). Additionally, the activity of Bacillus subtilis phage phi29 terminase requires a phage-encoded RNA molecule bound to its portal protein (8), and in vivo packaging of the E. coli phage T4 genome can only be completed with the participation of the phage-encoded endonuclease, gp49 (9). The general prevalence and importance of terminase cofactors is difficult to evaluate because few studies have addressed this issue.We recently reported that phage genomes often encode proteins possessing an HNH motif near their terminase genes (10). The HNH motif is ∼35 aa long, and is characterized by the presence of two highly conserved His residues and one Asn residue. These HNH motifs, as defined by the large (∼7,400-member) HNH Pfam (11) protein sequence family (PF01844), are often found in proteins that possess endonuclease activity, such as site-specific homing endonucleases (12, 13), colicins (14, 15), S pyocins (16), and restriction enzymes (17–19). HNH motif-containing proteins comprised of primarily an HNH motif as found in E. coli colicins, usually possess nonspecific endonuclease activity. Conversely, HNH motif-containing proteins may contain DNA-recognition domains in addition to the HNH motif and thus possess high sequence specificity, as found in the homing endonucleases.The frequent juxtaposition of HNH and phage terminase genes (10, 20) suggests a unique role for HNH proteins in the endonuclease and/or packaging activities of the terminases. To address this issue, we investigated the function of E. coli phage HK97 gp74, a 119-residue protein containing an HNH motif. The gene encoding gp74 is located at the extreme 3′ end of the mature linear HK97 genome, adjacent to the cos site. In both the lysogen and replicative form of the HK97 genome gene 74 is immediately adjacent to genes 1 and 2, which encode the small and large subunits of terminase (TerS and TerL), respectively. Whereas gp74 was previously found to possess endonuclease activity (10), its role in the HK97 replication cycle remained uncharacterized. In this study we used functional and bioinformatic analyses to investigate its function.     

The role of uninjured nerve in spinal nerve ligated rats points to an improved animal model of neuropathic pain.

L5 and L6 spinal nerve ligation (SNL) in rats leads to behavioral signs of neuropathic pain including mechanical allodynia. The purposes of this study were to investigate the role of the intact L4 spinal nerve in the development of mechanical allodynia following L5 and L6 SNL and, as a result, to develop a modified model of neuropathic pain. As a first set of experiments, in addition to tight ligation of the left L5 and L6 spinal nerves, the intact L4 spinal nerve was manipulated either (1) by gentle repeated stretching of the L4 spinal nerve immediately after L5 and L6 SNL or (2) by intermittent mechanical stimulation to the ipsilateral paw during the first week after SNL. Tactile sensitivity was measured by determining the foot withdrawal threshold before and after SNL. Mild irritation of L4 spinal nerve and application of mechanical stimuli to the ipsilateral paw significantly increased the development of mechanical allodynia after SNL. In a second set of experiments, SNL was produced by tightly ligating only the left L5 spinal nerve with or without a loop of 5-0 chromic gut placed loosely around the L4 spinal nerve. This additional L4 loop significantly increased long-lasting tactile sensitivity compared to L5 SNL alone. These results suggest that afferent activity of the intact L4 spinal nerve aids in the development of mechanical allodynia in the SNL model of neuropathic pain. The addition of a chromic gut loop around the intact L4 spinal nerve can augment the development of mechanical allodynia following SNL of L5. We propose this latter as a useful and practical animal model of neuropathic pain.