Nuclear protein kinase C isoforms and apoptosis

The process of apoptosis is regulated at multiple levels through phosphorylation by several different protein kinases. The protein kinase C (PKC) family of isozymes have been shown to exert both inhibitory and stimulatory influences on apoptosis. During the apoptotic process phosphorylative events are known to occur also at the nuclear level. Evidence suggests that PKC isoforms play a key role in some steps that lead to nuclear disassembly during the execution phase of apoptosis. This review highlights the recent progress made in determining the roles played by individual PKC nuclear isoforms in the control of apoptosis.     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a M(r) of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His(108), Asp(109), and His(168). We also identified Glu(142) as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.     

Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb,p107, and p130

Oxidative stress induces cell death and growth arrest. In this study, the regulation and the functional role of the retinoblastoma family proteins pRb, p107, and p130 in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2-treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb, and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed, it was found that inhibition of PP2A by SV40 small t over-expression prevented DNA synthesis inhibition induced by H2O2.     

Intracytoplasmic injection of frozen-thawed epididymal spermatozoa in a nonhuman primate model,the cynomolgus monkey (Macaca fascicularis)

Intracytoplasmic sperm injection (ICSI) with frozen-thawed epididymal spermatozoa was performed in the cynomolgus monkey (Macacafascicularis) to produce embryos in vitro. Eleven sexually mature females were hyperstimulated with an GnRH agonist (1.8 mg active triptorelin per 2 kg body weight), followed (2 weeks later) by rFSH (37.5 IU per 2 kg daily) for 12 days, and finally 1000 IU of hCG. Epididymal spermatozoa were collected from a single adult male monkey. The first stimulation cycle resulted in 90 oocytes; 70% of which were metaphase II (MII). Sixty-four percent of these MII oocytes were fertilized. Comparing ovarian response of five monkeys that underwent a second stimulation cycle there was an increase in oocyte quantity (13.2 versus 9.2 oocytes per monkey) but the percentage of MII oocytes remained the same at 58%. Fertilization and cleavage rates were also reduced but there was an increase in the number of embryos available for transfer. Overall, four monkeys became pregnant resulting in the birth of two healthy infants and two abortions. These findings show that ovarian stimulation by GnRH-rFSH did not compromise the developmental competence of the oocytes; effective combination of cryopreservation of epididymal spermatozoa and ICSI is possible in nonhuman primate reproduction, and thus has potential application in the conservation of highly endangered nonhuman primate species, and the cynomolgus monkey is a reliable biomedical research model to study the potential risks and benefits associated with assisted reproductive techniques prior to approval for clinical trials on humans.     

Vertebrae numbers of the early hominid lumbar spine

General doctrine holds that early hominids possessed a long lumbar spine with six segments. This is mainly based on Robinson's (1972) interpretation of a single partial Australopithecus africanus skeleton, Sts 14, from Sterkfontein, South Africa. As its sixth last presacral vertebra exhibits both thoracic and lumbar characteristics, current definitions of lumbar vertebrae and lumbar ribs are discussed in the present study. A re-analysis of its entire preserved vertebral column and comparison with Stw 431, another partial A. africanus skeleton from Sterkfontein, and the Homo erectus skeleton KNM-WT 15000 from Nariokotome, Kenya, did not provide strong evidence for the presence of six lumbar vertebrae in either of these early hominids. Thus, in Sts 14 the sixth last presacral vertebra has on one side a movable rib. In Stw 431, the corresponding vertebra shows indications for a rib facet. In KNM-WT, 15000 the same element is very fragmentary, but the neighbouring vertebrae do not support the view that it is L1. Although in all three fossils the transitional vertebra at which the articular facets change orientation seems to be at Th11, this is equal to a large percentage of modern humans. Indeed, a modal number of five lumbar vertebrae, as in modern humans, is more compatible with evolutionary principles. For example, six lumbar vertebrae would require repetitive shortening and lengthening not only of the lumbar, but also of the entire precaudal spine. Furthermore, six lumbar vertebrae are claimed to be biomechanically advantageous for early hominid bipedalism, yet an explanation is lacking as to why the lumbar region should have shortened in later humans. All this raises doubts about previous conclusions for the presence of six lumbar vertebrae in early hominids. The most parsimonious explanation is that they did not differ from modern humans in the segmentation of the vertebral column.     

A low-resolution 3D model of the tetrameric alcohol dehydrogenase from Sulfolobus solfataricus

We describe the computation of a model of the thermophilic NAD-dependent homotetrameric alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH). Modeling is based on the knowledge that each monomer contains two Zn ions with catalytic and structural function, respectively. In the database of known structures, proteins with similar functions are either dimers containing two zinc ions per monomer or tetramers with one zinc ion per monomer. In any case, the sequence identity of the target to the possible templates is low. A threading procedure is therefore developed which includes constraints taking into account residue conservation both at the zinc ion binding and at the monomer-monomer interaction sites in the tetrameric unit. The model is consistent with previously reported data. Furthermore, cross-linking experiments are described which support the computed tetrameric model.