Birth of a Western Lowland gorilla (Gorilla gorilla gorilla) following in vitro fertilization and embryo transfer

A 21-year-old multiparous female exhibiting 31–41 day menstrual cycles was given hFSH (225 IU/day, Metrodin 75, from cycle day 3 through 9 (menses = day 1) and hCG (10,000 IU, Profasi, on day 10 to stimulate follicular development. At 35 h after hCG, under isoflurane (AErrane) anesthesia, follicles were aspirated by controlled suction under transvaginal ultrasound guidance. Metaphase II oocyctes (n = 11) were placed in modified human tubal fluid (mHTF, 100 μl) medium under oil at 37°C in humidified 5% CO₂. Frozen semen, collected by voluntary ejaculation, was thawed (70°C H₂O bath, 6 sec), diluted slowly, centrifuged, and resuspended in mHTF, and 160,000 motile spermatozoa/ml were added at 6 h after oocyte recovery. At 21 h postinsemination (p.i.) eight oocytes were at the two-cell stage, five were cryopreserved, and three were cultured to the six- to eight-cell stage in mHTF with granulosa cells before transcervical uterine transfer at 47 h p.i. using a Teflon catheter. Micronized progesterone (400 mg/d) was orally administered for 10 weeks posttransfer (p.t.). Ultrasound examination revealed a single fetus at 15 weeks p.t., and unassisted delivery of a live 1.37 kg female infant occurred at 29 weeks. Am. J. Primatol. 41:247–260, 1997. © 1997 Wiley-Liss, Inc.     

Proteases of human brain

Growing appreciation of the multiple functions of proteolytic enzymes in intracellular protein degradation and post-translational modification, in the release of biologically active macromolecules and peptides from precursors and in cellular protein regulation and quality control has stimulated interest in proteases in neurobiology and neuropathology. In this article, the proteinases and peptidases thus far studied in the human central nervous system are reviewed with respect to their enzymology, anatomical and cytological distributions and contributions to neurological and psychiatric disease states. Though information concerning brain proteases in man is fragmentary, it suffices to establish the importance of these complex systems for advancing knowledge of human cerebral function in health and disease.The authors are privileged to submit this contribution for the Special Issue of Neurochemical Research honoring Professor K. A. C. Elliott—distinguished pioneer in neurochemistry and a cherished mentor and former colleague of one of us (AP).     

Inhibition of forskolin-stimulated cAMP formation in vitro by paraoxon and chlorpyrifos oxon in cortical slices from neonatal, juvenile, and adult rats

Parathion (PS) and chlorpyrifos (CPF) are organophosphorus insecticides, which elicit toxicity following biotransformation to the potent acetylcholinesterase inhibitors, paraoxon (PO) and chlorpyrifos oxon (CPO). Both oxons have also been shown to interact directly with muscarinic receptors coupled to inhibition of adenylyl cyclase. Immature animals are more sensitive than adults to the acute toxicity of PS and CPF but little is known regarding possible age-related differences in interactions between these toxicants and muscarinic receptors. We compared the inhibition of forskolin-stimulated cAMP formation by PO and CPO (1 nM-1 mM) in vitro in brain slices from 7-, 21-, and 90-day-old rats to the effects of well-known muscarinic agonists, carbachol and oxotremorine (100 microM). Both agonists inhibited cAMP formation in tissues from all age groups and both were more effective in adult and juvenile (20-26% inhibition) than in neonatal (12-13% inhibition) tissues. Atropine (10 microM) completely blocked agonist-induced inhibition in all cases. PO maximally inhibited (37-46%) cAMP formation similarly in tissues from all age groups, but atropine blocked those effects only partially and only in tissues from 7-day-old rats. CPO similarly inhibited cAMP formation across age groups (27-38%), but ATR was partially effective in tissues from all three age groups. Both oxons were markedly more potent in tissues from younger animals. We conclude that PO and CPO can directly inhibit cAMP formation through muscarinic receptor-dependent and independent mechanisms and that the developing nervous system may be more sensitive to these noncholinesterase actions.     

Cervical spine involvement in rheumatoid arthritis over time: results from a meta-analysis

IntroductionComplications in rheumatoid arthritis (RA) seem less common than they were years ago. The prevalence and progression of anterior atlantoaxial subluxations (aAASs), vertical subluxations (VSs), subaxial subluxations (SASs), and associated cervical myelopathy in RA over the past 50 years were determined.MethodsA literature search was performed by using Medline-OVID/EMBASE, PubMed, and Scopus (from 1960 to June 21, 2014). Prevalence studies were included if the sample size was at least 100 or the prevalence/progression of cervical subluxations was reported. Study quality was assessed by using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist. Prevalence of cervical subluxations was calculated for each study. Student’s t test and meta-regression were used to evaluate for significance.ResultsIn total, 12,249 citations were identified and 59 studies were included. The prevalence of aAAS decreased from 36% (95% confidence interval (CI) 30% to 42%) before the 1980s to 24% (95% CI 13% to 36%) in the 2000s (P = 0.04). The overall prevalence rates were 11% (95% CI 10% to 19%) for VS, 13% (95% CI 12% to 20%) for SAS, and 5% (95% CI 3% to 9%) for cervical myelopathy, and there were no significant temporal changes. Rates of progression of aAAS, VS, and SAS were 4, 6, and 3 lesions per 100 patients per year, respectively. The incidence of new or progressive cervical myelopathy was 2 cases per 100 patients with known cervical subluxations per year.ConclusionsSince the 1960s, only aAAS has decreased dramatically. It is still more than twice as common as VS or SAS. No temporal changes in the development of cervical myelopathy in affected patients with RA were noted. The progression rates of cervical subluxations and myelopathy were unchanged over time.     

Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5

Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine to generate 3-O-sulfated heparan sulfate (HS), which is a rare component in HS from natural sources. We previously reported that 3-O- sulfotransferase isoform 5 (3-OST-5) generates both an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpes simplex virus 1 glycoprotein D to serve as an entry receptor for herpes simplex virus. In this study, we characterize the substrate specificity of 3-OST-5 using the purified enzyme. The enzyme was expressed in insect cells using the baculovirus expression approach and was purified by using heparin-Sepharose and 3',5'-ADP- agarose chromatographies. As expected, the purified enzyme generates both an antithrombin binding site and a glycoprotein D binding site. We isolated IdoUA-AnMan3S and IdoUA-AnMan3S6S from nitrous acid-degraded 3-OST-5-modified HS (pH 1.5), suggesting that 3-OST-5 enzyme sulfates the glucosamine residue that is linked to an iduronic acid residue at the nonreducing end. We also isolated a disaccharide with a structure of DeltaUA2S-GlcNS3S and a tetrasaccharide with a structure of DeltaUA2S-GlcNS-IdoUA2S-GlcNH23S6S from heparin lyases-digested 3-OST-5-modified HS. Our results suggest that 3-OST-5 enzyme sulfates both N-sulfated glucosamine and N-unsubstituted glucosamine residues. Taken together, the results indicate that 3-OST-5 has broader substrate specificity than those of 3-OST-1 and 3-OST-3. The unique substrate specificity of 3-OST-5 serves as an additional tool to study the mechanism for the biosynthesis of biologically active HS.     

Purine Utilization by Klebsiella oxytoca M5al: Genes for Ring-Oxidizing and -Opening Enzymes

The enterobacterium Klebsiella oxytoca uses a variety of inorganic and organic nitrogen sources, including purines, nitrogen-rich compounds that are widespread in the biosphere. We have identified a 23-gene cluster that encodes the enzymes for utilizing purines as the sole nitrogen source. Growth and complementation tests with insertion mutants, combined with sequence comparisons, reveal functions for the products of these genes. Here, we report our characterization of 12 genes, one encoding guanine deaminase and the others encoding enzymes for converting (hypo)xanthine to allantoate. Conventionally, xanthine dehydrogenase, a broadly distributed molybdoflavoenzyme, catalyzes sequential hydroxylation reactions to convert hypoxanthine via xanthine to urate. Our results show that these reactions in K. oxytoca are catalyzed by a two-component oxygenase (HpxE-HpxD enzyme) homologous to Rieske nonheme iron aromatic-ring-hydroxylating systems, such as phthalate dioxygenase. Our results also reveal previously undescribed enzymes involved in urate oxidation to allantoin, catalyzed by a flavoprotein monooxygenase (HpxO enzyme), and in allantoin conversion to allantoate, which involves allantoin racemase (HpxA enzyme). The pathway also includes the recently described PuuE allantoinase (HpxB enzyme). The HpxE-HpxD and HpxO enzymes were discovered independently by de la Riva et al. (L. de la Riva, J. Badia, J. Aguilar, R. A. Bender, and L. Baldoma, J. Bacteriol. 190:7892-7903, 2008). Thus, several enzymes in this K. oxytoca purine utilization pathway differ from those in other microorganisms. Isofunctional homologs of these enzymes apparently are encoded by other species, including Acinetobacter, Burkholderia, Pseudomonas, Saccharomyces, and Xanthomonas.Purines and purine derivatives comprise a large portion of biomass and are involved in almost every step of life. Not only a major constituent of nucleic acids, they also are central to energy transfer and storage (ATP) as well as protein synthesis and signaling (GTP). Plants, animals, and many microorganisms use purines and purine derivatives to store and translocate nitrogen for assimilation or excretion (96).Salvage pathways operate to recycle purines, including hypoxanthine and xanthine, back into nucleoside pools (107). Additionally, some organisms can utilize purines as the sole source of nitrogen and carbon. Adenine and guanine are deaminated to form hypoxanthine and xanthine, respectively, which then are oxidized to form uric acid (urate at physiological pH) (Fig. ​(Fig.1).1). These oxidation steps are catalyzed by xanthine dehydrogenase, a well-studied molybdoflavoenzyme that is conserved from bacteria to humans (51). Two sequential ring-opening steps convert urate via allantoin to allantoate (Fig. ​(Fig.1).1). Subsequent steps, which comprise different pathways in different microorganisms (96), convert allantoate to ammonium, which is assimilated.Open in a separate windowFIG. 1.Purine ring oxidation and opening steps. The enzyme proposed to catalyze each step is shown. The K. oxytoca gene for adenine deaminase was not identified in this study. Dashed lines show reactions that can occur spontaneously.Some organisms express only the latter portion of the purine utilization pathway and cannot use purines or urate as sole sources of nitrogen. For example, Escherichia coli K-12 can use allantoin and its catabolites as the sole nitrogen source, albeit only under anaerobic conditions (21). Saccharomyces cerevisiae uses allantoin as a nitrogen storage compound (17). However, the complete pathway is present in other bacterial and fungal species, including Bacillus subtilis (84) and Aspergillus nidulans (83).Molybdoenzymes (excepting dinitrogenase) contain the molybdenum cofactor Mo-molybdopterin (42). Thus, mutations in genes for molybdenum cofactor biosynthetic enzymes (mol genes in bacteria and cnx in A. nidulans) confer pleiotropic phenotypes: these mutants can utilize neither nitrate nor purines, due to lack of the molybdoenzymes nitrate reductase and xanthine dehydrogenase (74). We previously reported that Klebsiella oxytoca mol mutants cannot assimilate nitrate but can utilize xanthine as the sole nitrogen source (32). This suggested, as one possibility, that K. oxytoca uses a molybdenum-independent enzyme in place of conventional xanthine dehydrogenase. Results reported here demonstrate that this is correct, as insertion mutants blocked specifically in xanthine and hypoxanthine utilization define the structural genes for an apparent two-component Reiske nonheme iron oxygenase.Here, we report analysis of 12 genes whose products catalyze conversion of purines to allantoate. Our investigation of the remaining genes, whose products catalyze allantoate utilization, is ongoing. Results show that several steps in the overall pathway are catalyzed by previously undescribed enzymes.While this paper was in review, the paper by de la Riva et al. (24), describing the hpxDE, hpxR, hpxO, and hpxPQT genes from Klebsiella pneumoniae W70, was posted in the “JB Accepts” section of the Journal of Bacteriology online edition. Results and conclusions concerning these seven genes are congruent between the two studies.(Some of the work presented here was submitted by Danielle Carl in 1994 as part of an undergraduate thesis to the Cornell University Division of Biological Sciences Honors Program.)