A procedure to secure a jugular vein catheter system onto the neck of the hamster

We describe a procedure to secure a jugular vein catheter system at the dorsal nape of the neck in the hamster. An 8-cm piece of silicone tubing is connected with a 2.6 cm L-shaped metal tubing which is embedded in prosthetic material. The prosthetic material is placed underneath the neck skin of the hamster and keeps the metal end of the catheter system in a sturdy, upright position.     

The effects of cisplatinum and vincristine on peripheral nerve regeneration

Current treatment modalities for extremity sarcoma often include tumor extirpation plus neoadjuvant therapy. Limb-sparing surgery may require reconstruction of critical nerve defects. Neurotoxic side effects from adjuvant chemotherapy have been reported and raise concerns regarding the effects of chemotherapy on nerve regeneration. In an attempt to define the effects of adjuvant chemotherapy on peripheral nerve regeneration, cisplatin and vincristine were administered to rats following isografting of the posterior tibial nerve. Parameters used to assess peripheral nerve regeneration included walking track analysis and histomorphology. Sixty 250-g Sprague-Dawley rats were randomly allocated into one of three treatment groups. Each animal underwent a 15-mm reversed interposition nerve isograft from 30 donor rats into the right posterior tibial nerve. Ten animals served as control. The remaining animals were divided into two groups of 25 animals each. One group received cisplatin (75 mg/m2) and the other group received vincristine (1 mg/m2). Chemotherapy was administered at 4-week cycles for a total of six cycles (24 weeks). Walking track analysis was performed monthly. Nerve specimens were harvested from the grafted segment and the distal posterior tibial nerve for histomorphology. Walking track analysis demonstrated no statistical difference in print length between the control and chemotherapeutic groups at the conclusion of the study. The number of axons per square millimeter and nerve fiber density were not statistically different between control and chemotherapeutic groups. In the rodent posterior tibial nerve model, postoperative adjuvant therapy does not significantly alter functional outcome in peripheral nerve regeneration. The practice of immediate nerve grafting after tumor extirpation, despite planned postoperative chemotherapy, is supported.     

The synthesis and SAR of 2-arylsulfanyl-phenyl piperazinyl acetic acids as glyT-1 inhibitors

Elevation of glycine levels and activation of the NMDA receptor by inhibition of the glycine transporter 1 (GlyT-1) is a potential strategy for the treatment of schizophrenia. A novel series of GlyT-1 inhibitors have been identified containing the 2-arylsulfanyl-phenylpiperazine motif. The most prominent member of this series, (R)-4-[5-chloro-2-(4-methoxy-phenylsulfanyl)-phenyl]-2-methyl-piperazin-1-yl-acetic acid (31) is a potent glycine transporter-1 inhibitor (IC(50)=150 nM), which elevated glycine levels in rat ventral hippocampus as measured by microdialysis in vivo at doses of 1.2-4.6 mg/kg s.c.     

Prevalence of major depression one year after predictive testing for Huntington's disease

Psychiatric hospitalizations, completed suicides, and suicide attempts are rare after predictive testing for Huntington's disease (HD). Case studies have shown that major depression can be a consequence of being tested, although no studies have shown how common this is. The present study evaluated the prevalence of major depression during the first year after disclosure. We conducted retrospective data and chart reviews of 153 persons (50 testing positive, 103 testing negative) evaluated every 3 months for depression. There was no significant baseline difference in the percentage of "positives" and "negatives" who had pre-testing major depressive episodes (14% vs. 12%, respectively). A senior psychiatrist reviewed data from the Schedule for Affective Disorders and Schizophrenia-Change Version, from the Beck Depression Inventory, and from clinical notes for every follow-up contact completed. The 1-year prevalence of major depression among positives was 6.0%, compared to 3.0% among negatives (p = 0.30), and an estimated 3% population prevalence. One-year prevalence of clinically significant depressive symptoms, whether or not major depression was diagnosed, was 20.0% in positives and 12.6% in negatives (p = 0.17). Although not statistically significant, depressive symptoms and major depression occurred more frequently among those who tested positive. Despite some evidence to the contrary, including our own studies, a positive predictive test for HD is not psychologically benign. Clinical testing programs should assess patients for depressive symptoms after testing, and patients with clinically significant complaints should be referred to a mental health professional.     

B cell development leads off with a base hit: dU:dG mismatches in class switching and hypermutation

The mechanisms underlying somatic hypermutation (SHM) and class switch recombination (CSR) have been the subject of much debate. Recent studies from the Neuberger and Honjo labs have lent insight into these distinct processes, and we discuss a new, comprehensive model for how AID, uracil DNA glycosylase (UNG) and the mismatch repair system function in both SHM and CSR.     

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).     

Structure/function relationships responsible for coenzyme specificity and the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).