Crystallization and preliminary diffraction analysis of Ca2+-calmodulin-drug and apocalmodulin-drug complexes

Ca²⁺-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3”-(β-chloroethyl)-2”,4”-dioxo-3,5”-spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4-di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 Å and differ in unit cell parameters from each other as well as from crystals of Ca²⁺-calmodulin or Ca²⁺-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca²⁺-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca²⁺-free calmodulin crystals diffracting beyond 2.5 Å resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca²⁺-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies. © 1997 Wiley-Liss Inc.     

Purification and analysis of a 5kDa component of enamel matrix derivative

High performance liquid chromatography (HPLC) methods were used to analyse a 5kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm x 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5kDa component. From 1200 mg of EMD (of which 9% is the 5kDa component), approximately 65 mg of lyophilised 5kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.     

Neural tube defects, micronutrient deficiencies, and Helicobacter pylori: a new hypothesis

BACKGROUND: Previous findings for the Texas Neural Tube Defects Project suggested that while maternal access to nutrients is adequate, bioavailability of nutrients to the fetus is compromised in NTD-affected pregnancies. Helicobacter pylori could cause nutrient loss to the fetus. Folate, B12, and ferritin are depleted in H. pylori infection; these same deficiencies are related to NTD risk. METHODS: Using H. pylori IgG ELISA Test System, we tested for H. pylori serum antibodies in participants in the population-based case-control study component of the Texas Neural Tube Defect Project conducted along the Texas-Mexico border. Case-women had pregnancies affected by NTD (anencephalus, spina bifida, encephalocele) and resided and delivered in one of the 14 Texas-Mexico border counties from 1995 through 2000. Control-women were study area residents delivering normal live births during the same period. RESULTS: Of 225 case- and 378 control-women, 103 cases and 156 controls provided questionnaire and H. pylori antibody data. H. pylori seropositivity was modestly associated with NTD-affected pregnancies (OR 1.4; 95% CI: 0.8-2.4). ORs of 2.0 or greater were seen in women younger than age 25 and with less than 7 years education. CONCLUSIONS: Our findings intimate that H. pylori could play a role in NTD causation in certain populations. While results did not provide compelling support for this proposal, subgroup findings prompt us to advocate an evaluation of this hypothesis in developing nations among populations with higher prevalence of H. pylori, marginal nutrient intake, and young childbearing age.     

An internal signal sequence directs intramembrane proteolysis of a cellular immunoglobulin domain protein

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins.     

Thiopurine S-Methyltransferase Gene Polymorphisms in a Healthy Slovak Population and Pediatric Patients with Inflammatory Bowel Disease

Thiopurine methyltransferase (TPMT) is a key component in thiopurine metabolism. There is an insufficient evidence about the distribution of the genotype frequencies of TPMT variants and frequencies of TPMT alleles associated with intermediate and deficient activity in a healthy Slovak population and pediatric patients with inflammatory bowel disease (IBD). TPMT variant alleles (*1,*2, *3A, *3B, and *3C) were determined in 114 children treated for IBD and in 281 healthy volunteers. Mutant alleles were present in 9/114 (7.89%) in the IBD patients and in 23/281 (8.19%) of probands. The distribution of the most frequent variants of TPMT gene was similar in a healthy population and patients with IBD.