Thalassaemia,iron, and pregnancy.

Haematological values of 35 pregnant women with beta-thalassaemia trait were followed during pregnancy. The discriminant function, calculated from haematological indices, was of no value in diagnosing beta-thalassaemia trait during pregnancy. Initially patients were given iron supplements only if the serum iron and total iron binding capacity levels indicated iron deficiency, but bone marrow biopsies performed in the first 22 patients at 32 weeks indicated deficient iron stores. These patients were therefore given iron irrespective of their serum iron level. All subsequent patients with beta-thalassaemia were also put on iron routinely at booking. Retrospectively the patients were divided into two groups. Patients in group 1 (18 patients) had received iron for less than 12 weeks, and their haemoglobin levels fell significantly during pregnancy (P less than 0-001). Haemoglobin levels in 16 patients who had received iron for more than 12 weeks (group 2), however, did not fall significantly during pregnancy (P less than 0-6). It is suggested (contrary to common practice) that patients with beta-thalassaemia trait should be given iron supplements during pregnancy. Serum folate and vitamin B12 levels did not change significantly in these patients and there was no increase in the incidence of maternal or fetal complications.     

Production and characterisation of a monoclonal antibody to progesterone and its effect on fertility

A monoclonal antibody reacting with progesterone has been raised by fusion of mouse myeloma cells (SP20) and splenocytes of BALB/c mice hyperimmunized with 4-pregnane 3,20 dione conjugated to bovine serum albumin. Association constant of this antibody for binding with progesterone was 0.22 x 10(9) l/mole. The antibody was highly specific for progesterone. A single ip injection of this antibody brought about an antifertility effect which is influenced by genotype. Antibody treatment brought about a significant decrease in the fetal weight and a slight decrease in the plasma progesterone levels. The antifertility effect could be reversed only up to day 3 by exogenous administration of progesterone.     

Innervation of radicular and extraparenchymal arteries of spinal cord

Summary The perivascular innervation of extraparenchymal arteries of spinal cord and the radicular arteries was examined using histochemical and immunohistochemical technics in monkey. The radicular and the extraparenchymal arteries of spinal cord were found to be invested with adrenergic, neuropeptide Y, vasoactive intestinal peptide, substance P and calcitonin gene-related peptide containing nerve fibres. The pattern of arrangement of fibres differed among the various fibre types. SP-and CGRP-containing fibres were less in density as compared to other nerve plexus. There was no difference in density of an individual type of nerve fibre in arteries of different cord segments or between the radicular arteries from different levels. The study reveals the existence of a comprehensive perivascular adrenergic and peptidergic innervation of spinal cord arterial system, with a possible role in neurogenic regulation of spinal cord circulation.     

Hydrogen sulfide up-regulates substance P in polymicrobial sepsis-associated lung injury

Hydrogen sulfide (H2S) has been shown to induce the activation of neurogenic inflammation especially in normal airways and urinary bladder. However, whether endogenous H2S would regulate sepsis-associated lung inflammation via substance P (SP) and its receptors remains unknown. Therefore, the aim of the study was to investigate the effect of H2S on the pulmonary level of SP in cecal ligation and puncture (CLP)-induced sepsis and its relevance to lung injury. Male Swiss mice or male preprotachykinin-A gene knockout (PPT-A-/-) mice and their wild-type (PPT-A+/+) mice were subjected to CLP-induced sepsis. DL-propargylglycine (50 mg/kg i.p.), an inhibitor of H2S formation was administered either 1 h before or 1 h after the induction of sepsis, while NaHS, an H2S donor, was given at the same time as CLP. L703606, an inhibitor of the neurokinin-1 receptor was given 30 min before CLP. DL-propargylglycine pretreatment or posttreatment significantly decreased the PPT-A gene expression and the production of SP in lung whereas administration of NaHS resulted in a further rise in the pulmonary level of SP in sepsis. PPT-A gene deletion and pretreatment with L703606 prevented H2S from aggravating lung inflammation. In addition, septic mice genetically deficient in PPT-A gene or pretreated with L703606 did not exhibit further increase in lung permeability after injection of NaHS. The present findings show for the first time that in sepsis, H2S up-regulates the generation of SP, which contributes to lung inflammation and lung injury mainly via activation of the neurokinin-1 receptor.     

Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.     

Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel

P450BM-3 is an extensively studied P450 cytochrome that is naturally fused to a cytochrome P450 reductase domain. Crystal structures of the heme domain of this enzyme have previously generated many insights into features of P450 structure, substrate binding specificity, and conformational changes that occur on substrate binding. Although many P450s are inhibited by imidazole, this compound does not effectively inhibit P450BM-3. Omega-imidazolyl fatty acids have previously been found to be weak inhibitors of the enzyme and show some unusual cooperativity with the substrate lauric acid. We set out to improve the properties of these inhibitors by attaching the omega-imidazolyl fatty acid to the nitrogen of an amino acid group, a tactic that we used previously to increase the potency of substrates. The resulting inhibitors were significantly more potent than their parent compounds lacking the amino acid group. A crystal structure of one of the new inhibitors bound to the heme domain of P450BM-3 reveals that the mode of interaction of the amino acid group with the enzyme is different from that previously observed for acyl amino acid substrates. Further, required movements of residues in the active site to accommodate the imidazole group provide an explanation for the low affinity of imidazole itself. Finally, the previously observed cooperativity with lauric acid is explained by a surprisingly open substrate-access channel lined with hydrophobic residues that could potentially accommodate lauric acid in addition to the inhibitor itself.     

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K_d ～20 nM) via the common interacting interface (residues 312–349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CΔ78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CΔ78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.