Pyroglutamyl-peptidase I: cloning,sequencing, and characterisation of the recombinant human enzyme

Pyroglutamyl-peptidase I (EC 3.4.19.3) is well known from bacteria and archaea, but has not previously been cloned or sequenced from any vertebrate. We describe the cloning and sequencing of the human (AJ278828) and mouse (AJ278829) forms of pyroglutamyl-peptidase I. The deduced amino acid sequences each consist of 209 residues and show approximately 30% identity with bacterial forms of the enzyme. They show clear homology to the enzyme from prokaryotes and place the mammalian forms of the enzyme in peptidase family C15 of the MEROPS database. The catalytic residues Glu81, Cys144, and His166 in the enzyme from Bacillus amyloliquefaciens are all conserved in the human sequence. A simple cartoon model of the human protein was constructed on the basis of the published crystal structures of pyroglutamyl-peptidase I forms from Thermococcus litoralis and B. amyloliquefaciens. The human enzyme was expressed by use of a baculovirus vector in Spodoptera frugiperda cells. The recombinant protein was enzymatically active and had properties similar to those described for the naturally occurring mammalian enzyme. Gel-filtration chromatography of the active enzyme gave a molecular mass of about 24kDa, showing that the enzyme is active as the monomer. This contrasted with indications that the prokaryotic enzymes may be tetrameric. Recombinant human pyroglutamyl-peptidase I was active on pGlu-aminomethylcoumarin in the range pH 6-9, with maximal activity being seen at pH 7.0-8.5; it showed an absolute requirement for a thiol-reducing agent. In crude preparations, the enzyme was completely stable for 90 min at 50 degrees C. The enzyme was inhibited by transition metal ions including Ni(2+), Zn(2+), and Cu(2+), and by sulfhydryl-blocking agents. Reversible inhibition was seen with 2-pyrrolidone (K(i)=50 microM), and surprisingly, with N-ethylmaleimide (K(i)=30 microM).     

Finger length and distal finger extent patterns in humans

The fingers in the adult human hand differ in length and in distal extent. The literature agrees that in the clear majority of males, the distal extent of the ring finger tends to be relatively greater (using the middle finger as standard) than the index finger. However, the results for females vary considerably, with some studies reporting that females show a similar pattern to that of males, while others suggest that the prevalence of a longer index finger is relatively or absolutely more common in females. We provide a review of the literature, and a set of data for both finger length and distal fingertip extent of the finger for a contemporary cohort of young adult females and males (n = 502). Finger length measures favor the ring finger of both sexes, with smaller between-finger differences for females than for males. However, while the distal fingertip extent favors the ring finger of both hands in males, in females the left hand shows no significant differences, and the right hand shows a small index finger advantage. Thus, the sexual dimorphism in finger measures is more strongly expressed in the distal extent of fingertips than in the length of fingers. The sex differences in distal fingertip extent derive from the index finger only, with a lesser distal extent of the index finger, relative to the middle finger, in males than in females.     

Glucocorticoid-induced apoptosis in early B cells from human bone marrow

The sensitivity of normal human lymphoid precursor cells to glucocorticoid-induced apoptosis is a subject of controversy. The in vitro response of cells of the B lineage (CD19(+)) from the marrow of 22 adult subjects to glucocorticoids was evaluated herein using both natural steroids and dexamethasone (Dex). When exposed to 1 micro M Dex, 32% of the subjects exhibited high losses of CD19(+) B cells in the range of 45%. The remaining subjects exhibited more modest losses in CD19(+) cells of 26%-40%. Surprisingly, cortisol, a naturally produced glucocorticoid, produced B lineage losses nearly equivalent to Dex, which reached maximum by 12 hr. It was subsequently noted that the variances in losses of CD19(+) cells among the subjects correlated closely with the proportion of early CD10(+) CD19(+) B cells present in the initial population. The latter cells exhibited a high degree of sensitivity to glucocorticoids, with losses of 60%-80% noted. Mature B cells bearing IgD, on the other hand, were fairly resistant to glucocorticoids. Merocyanine 540, a membrane dye that fluoresces in the disordered membrane of apoptotic cells, confirmed that early or progenitor B cells in human bone marrow were indeed undergoing glucocorticoid-induced apoptosis, which could be blocked by the glucocorticoid antagonist RU38486. These data provide evidence that human marrow B cells, especially early B-cell progenitors, are quite sensitive to glucocorticoids and readily undergo apoptosis within a few hours of exposure to the steroids.     

Superparasitism and sex ratio in the solitary parasitoidEpidinocarsis lopezi

Highly variable sex ratios are found in the solitary parasitoidEpidinocarsis lopezi, both in the field and in a mass-rearing situation. Superparasitism is one of a number of factors which can influence sex ratios in parasitoid Hymenoptera. We show that superparasitism inE. lopezi is common in the field. Sex allocation decisions when parasitizing unparasitized hosts are not different from those with parasitized hosts; neither does differential mortality occur between the sexes in superparasitized hosts. Therefore superparasitism does not contribute to the variable sex ratio ofE. lopezi. Both the occurrence of superparasitism and the sex produced when ovipositing are shown to be functional forE. lopezi.     

Temperature biofeedback and relaxation training in the treatment of migraine headaches

In a previous controlled group outcome study, a comparison of temperature biofeedback with progressive relaxation indicated that relaxation training was more effective in reducing migraine headache activity at the end of treatment. However, follow-up data obtained at 1, 2, and 3 months after the completion of treatment showed no difference between the two groups on any dependent measure. In the current study, 18 of 26 subjects who completed treatment in the original investigation collected headache data and completed a headache questionnaire I year subsequent to the conclusion of treatment in order to evaluate the long-term effectiveness of the two treatments. The results indicated that gains achieved in the reduction of headaches during both treatments were maintained at a 1-year follow-up. With the exception of medication consumption (for which relaxation training led to better long-term results) the 1-year follow-up data reveal no differential efficacy for temperature biofeedback or progressive relaxation in treating migraine headaches.     

siRNA relieves chronic neuropathic pain

Double stranded, short interfering RNAs (siRNA) of 21–22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X₃, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X₃ siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X₃ mRNA expressed in dorsal root ganglia, and P2X₃ protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.     

Induction of Th1-type immunity and tumor protection with a prostate-specific antigen DNA vaccine

Prostate specific antigen (PSA) is a serum marker that is widely used in the detection and monitoring of prostate cancer. Though PSA is a self-antigen, T cell responses to PSA epitopes have been detected in healthy men and prostate cancer patients, suggesting it may be used as a target for active immunotherapy of prostate cancer. A PSA DNA vaccine (pPSA) was evaluated in mice and monkeys for its ability to induce antigen-specific immune responses. Mice immunized intradermally with pPSA demonstrated strong PSA-specific humoral and cellular immunity. The anti-PSA immune responses were skewed toward Th1, as shown by high IFN and IL-2 production. The immune response was sufficient to protect mice from challenge with PSA-expressing tumor cells. Tumor protection was durable in the absence of additional vaccination, as demonstrated by protection of vaccinated mice from tumor rechallenge. Furthermore, pPSA vaccination induced PSA-specific antibody titers in male cynomolgus monkeys, which express a closely related PSA gene. These results demonstrate that vaccination with pPSA may be able to break tolerance and can induce an immune response that mediates tumor protection.     

Diacylglycerol-activated Hmunc13 serves as an effector of the GTPase Rab34

Hmunc13 is a cytosolic diacylglycerol (DAG)-binding protein, which is upregulated in renal cortical tubule and mesangial cells by hyperglycemia. In response to DAG activation, hmunc13 translocates to the Golgi. To investigate how this may relate to its function, we used a bacterial two-hybrid screen to look for hmunc13-interacting proteins. Full-length Rab34 was specifically isolated from a human kidney cDNA library. Co-expression of the two proteins confirmed Rab34 as a Golgi-associated protein, which was immunoprecipitated from cell lysates by hmunc13. Glutathione S-transferase fusion proteins of WT, Q111L (GTP bound), and T66N (GDP bound) mutants were created, and their GTP-binding activity verified by radioactive overlay assay. Binding of hmunc13 was observed with Q111L, barely detectable with T66N and enhanced with Rab34WT loaded with GTPgammaS compared with GDP loaded. Deletion of munc homolgy domain (MHD)-2, eliminated the hmunc13/Rab34 interaction. The Q111L mutant localized to the Golgi apparatus, but T66N was cytosolic. Localization of both mutants and Rab34WT was unchanged by DAG activation. The data suggest that DAG activation of hmunc13 causes it to be translocated to the Golgi, where it binds to GTP-bound Rab34 via MHD-2. Because Rab34 is known to regulate intracellular lysosome positioning, we propose that hmunc13 serves as an effector of Rab34, mediating lysosome-Golgi trafficking.