Studies on Bacillus stearothermophilus. Part 1. Transformation of progesterone to a new metabolite 9,10-seco-4-pregnene-3,9,20-trione

When Bacillus stearothermophilus, a thermophilic bacterium isolated from the Kuwaiti desert, was incubated with exogenous progesterone for 24 h, three monohydroxylated metabolites were produced. 20α-Hydroxyprogesterone was the major metabolite produced in 60.8 relative percentage yield. The other two monohydroxylated metabolites were identified as 6β-hydroxyprogesterone and the rare 6α-hydroxyprogesterone in 21.0 and 13.6 relative percentage yields, respectively. A new metabolite 9,10-seco-4-pregnene-3,9,20-trione was isolated in 3.7 relative percentage yield. All metabolites were purified by preparative TLC and HPLC followed by their identification using ¹H, ¹³C NMR and other spectroscopic data.     

Hepatitis B virus surface antigen assembly function persists when entire transmembrane domains 1 and 3 are replaced by a heterologous transmembrane sequence

Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.     

Studies on Bacillus stearothermophilus. Part IV. Influence of enhancers on biotransformation of testosterone

The impact of chemical enhancers on the biotransformation of testosterone has been exploited. Application of crude cell concentrates to produce Bacillus stearothermophilus-mediated bioconversion of testosterone at 65 degrees C for 72 h has been examined. After incubation, the xenobiotic substrate was added to the concentrated whole cell suspensions. The enhancer molecules were included in the whole cell suspension. The resultant products, after extraction into an organic solvent, were purified by thin layer chromatography and identification was carried out through spectroscopic data. Five steroid metabolites 9,10-seco-4-androstene-3,9,17-trione, 5alpha-androstan-3,6,17-trione, 17beta-hydroxy-5alpha-androstan-3,6-dione, 3beta,17beta-dihydroxyandrost-4-ene-6-one and 17beta-hydroxyandrost-4,6-diene-3-one were identified as biotransformation products of testosterone. A possible biosynthetic route for these bioconversion products is postulated.     

Increased brain uptake and brain to blood efflux transport of 14C-GABA in spontaneously hypertensive rats

The brain uptake and brain to blood efflux transport of (14)C-GABA were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats using 20 min bilateral in situ brain perfusion in rats anesthetized using urethane. The volume of distribution (Vd) of (14)C-GABA into cerebrospinal fluid (CSF) and brain regions (cortex, diencephalon, cerebellum, and brain stem) was significantly greater in SHR than in the corresponding regions in WKY rats (p<0.05). The estimated Vd value of (14)C-GABA in CSF of SHR was 3.4 fold greater than that in WKY. Also compared to WKY, the Vd of (14)C-GABA into cerebellum and cortex of SHR was 15.3 fold and 19.4 fold greater, respectively. Although the study of blood-brain barrier (BBB) integrity using (3)H-mannitol revealed increased paracellular permeability at the brain capillaries of SHR when compared to WKY rats, this was found to be only partially responsible for the increased (14)C-GABA uptake. The study of brain to blood efflux transport of (14)C-GABA (after loading of brain with (14)C-GABA by vascular perfusion) revealed that the half-time of elimination was significantly shorter in SHR (5.35+/-0.66 min) than in WKY rats (14.83+/-1.94 min), (p<0.001). HPLC analysis revealed that GABA concentrations in brain extracts and CSF of SHR were similar to those in WKY rats (p>0.05). The faster efflux in SHR might be, at least partially, responsible to compensate for increased uptake of this neurotransmitter and to preserve the protective function of BBB towards GABA. The protective function of the BCSFB towards GABA appears to be also preserved, since systemic infusion of GABA within a wide range of administered doses (0.004-5.00 mg/kg) produced an increase in GABA CSF concentration from around 0.5 microM to only 11 microM, and the obtained pattern of CSF GABA concentrations under these conditions did not differ between SHR and WKY rats, as revealed by HPLC.     

Ischemic preconditioning is not additive to preservation with hypothermia or crystalloid cardioplegia in the globally ischemic rat heart

The aim of this study was to evaluate the additive protective efficiency of ischemic preconditioning when used in combination with conventional clinically relevant cardioprotective methods of hypothermia or hypothermic cardioplegia during sustained global ischemia.Isolated rat hearts were aorta-perfused with Krebs-Henseleit buffer and were divided into six groups (n = 10 each). Group I: Ischemia at 34°C for 60 min; Group PC+I: preconditioned (PC) ischemia at 34°C, 2 episodes of 5 min ischemia and 10 min reperfusion at 34°C followed by I; Group HI: hypothermic ischemia at 10°C for 60 min; Group PC+HI: preconditioned (PC) hypothermic ischemia, 2 episodes of 5 min ischemia and 10 min reperfusion at 34°C followed by HI; Group CPL+HI: single dose of 'Plegisol' cardioplegia followed by HI; Group PC+CPL+HI: preconditioned hypothermic cardioplegia, followed by CPL+HI. At the end of 60 min ischemia, all the hearts were reperfused at 34°C for 30 min when post-ischemic recovery in left ventricular contractile function and coronary vascular dynamics was computed and compared.There was a significant depression in the post-ischemic recovery of developed pressure (P_max), positive derivative of pressure (+dp/dt), negative derivative of pressure (-dp/dt) and heterometric autoregulation (HA) of contractile force in all the groups, with no major differences between the groups. Left ventricular end-diastolic pressure (LVEDP) was significantly elevated after I at 34°C. Preconditioning (PC+I) prevented the rise in the LVEDP and this was accompanied by a significant reduction in the release of purine metabolises in the coronary effluents, particularly adenosine, during the immediate reperfusion period. Hypothermia (HI) provided essentially the same level of metabolic and mechanical preservation as offered by PC+I. Combination of hypothermia with preconditioning (PC+HI) or cardioplegia (PC+CPL+HI), did not further enhance the preservation. Post-ischemic recovery in the regional contractile function (segment shortening, %SS) followed nearly identical pattern to global (P_max) recovery. Post-ischemic recovery in coronary flow (CF) was significantly reduced and coronary vascular resistance (CVR) was significantly increased in all the groups. Myogenic autoregulation (transient and sustained) was generally enhanced indicating increased vascular reactivity. Preconditioning did not alter the time-course of these changes.Preconditioned ischemia (34°C) preserved left ventricular diastolic functions and prevented the contracture development after sustained ischemia reperfusion at 34°C. This protective effect of preconditioning was possibly mediated by the reduction in the breakdown of purine metabolises. Hypothermia alone or in combination with crystalloid cardioplegia prevented the irreversibility of the ischemic injury but produced contractile and vascular stunning which was not improved by ischemic preconditioning. The results of this study indicate that preconditioning when combined with hypothermia or hypothermic cardioplegia offered no significant additional protection.     

Autosomal recessive “uncomplicated” profound childhood deafness in an arabic family with high consanguinity

Summary An Arabic family has been studied in our genetic counselling centre. This family included 13 cases of heritable uncomplicated profound childhood deafness. All of these were the offspring of first cousin (F.C.) marriages. The inheritance pattern was consistent with autosomal recessive (A.R.) determination of the deafness and all the affected children studied showed identical phenotypic expression.     

Studies on purine enzymes in experimental colitis

Although the role of adenosine deaminase (ADA), adenylate deaminase (AMP-DA), purine nucleoside phosphorylase (PNP) is well documented in gastric and intestinal carcinoma, their role in inflammatory bowel diseases remains unknown. In the present study, we investigated the profile of these enzymes in blood and intestinal tissues during colitis. Colitis induced in Wistar rats by acetic acid was monitored by a marker enzyme myeloperoxidase (MPO). The tissue levels of MPO increased on 1, 2, 5 and 6 days post-administration (PA) of acetic acid and declined to the control levels by day 7 PA. In parallel the blood levels of ADA and AMP-DA decreased on days 1, 2 and 5 without any significant change on days 6 and 7 PA. Similar observations were recorded for these enzymes in the cytosolic extracts of colonic tissue specimens. In contrast, PNP remained unaltered in both blood and tissue samples. These findings suggest an inverse-relationship between inflammation and purine deaminases in both blood and tissues.     

Studies on Bacillus stearothermophilus. Part II. Transformation of progesterone

Bacillus stearothermophilus, a thermophilic bacterium isolated from Kuwaiti desert, when incubated with exogenous progesterone for 10 days at 65 degrees C produced two new dihydroxy isomers of progesterone, and two known compounds, 5 alpha-pregnane-3,6,20-trione and 6-dehydroprogesterone, along with the earlier reported monohydroxylated metabolites and a B-Seco compound. The two new dihydroxy compounds were identified as 6 alpha,20 alpha-dihydroxyprogesterone and 6 beta,20 alpha-dihydroxyprogesterone. These metabolites were purified by TLC and HPLC followed by their identification through 1H, 13C NMR and other spectroscopic data.     

In vivo inhibition of cyclooxygenase-2 by a selective phosphorothioated oligonucleotide

Inhibition of cyclooxygenase-2 (cox-2) is considered to be anti-inflammatory, whereas inhibition of the constitutive isozyme cox-1 causes renal and gastrointestinal toxicity. Therefore, to achieve an optimal anti-inflammatory effect, an inhibitor should be cox-2 selective without inhibiting cox-1. For this purpose, 10 different cox-2-selective phosphorothioated oligonucleotides (S-oligos) were tested to inhibit the cox-2 enzyme selectively in vivo. An aqueous solution of these S-oligos (3 mg/kg body weight) was injected intraperitoneally (i.p.) into male Sprague-Dawley rats with colitis induced by trinitrobenzene sulfonic acid (TNBS). The colonic levels of cox-2 protein, mRNA, myeloperoxidase (MPO), and prostaglandin E2 (PGE2) were increased significantly on day 1 and remained significantly elevated until day 7 post-TNBS administration, whereas cox-1 remained unaltered. Two S-oligos were found to be effective in reducing the level of cox-2 protein selectively without any effect on the cox-1. The effective S-oligo, but not the mismatched control oligo, reduced the tissue levels of PGE2 and MPO activity significantly. The effective S-oligo reduced the level of cox-2 but not the cox-1 mRNA significantly, whereas a mismatched or a sense control oligo did not affect the levels of these isoforms. M-fold analysis demonstrated extensive secondary structure formation in the cox-2 mRNA. These findings demonstrate that only a few selected sites in the cox-2 target mRNA are accessible in vivo, probably because of the presence of secondary structures. Suppression of cox-2 protein, PGE2, and MPO activity by the S-oligo might prove to be an anti-inflammatory property.     

Severe short-limb dwarfism resembling Grebe chondrodysplasia

Summary A severe, nonlethal short-limb bone dysplasia is described in two unrelated patients. The disorder is characterized by a peculiar facial appearance, rib anomalies and severe shortness and distortion of individual long bones, notably the humeri, tibiae, fibulae, metapodia and phalanges with marked irregularity and asymmetry of bone changes. The condition is differentiated from other bone dysplasias with extreme limb shortness, in particular Grebe chondrodysplasia.