Stably transfected Jurkat T cells were produced in which Bax expression is inducible by muristerone A. The cell death resulting from induction of the overexpression of Bax was prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The caspase-3 inhibitor Z-Asp-Glu-Val aspartic acid fluoromethylketone (Z-DEVD-FMK) had no effect on the loss of viability. The MPT was measured as the CyA plus ArA-preventable loss of the mitochondrial membrane potential (DeltaPsim). The MPT was accompanied by the release of cytochrome c from the mitochondria, caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. Z-DEVD-FMK had no effect on the loss of DeltaPsim and the redistribution of cytochrome c but did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. It is concluded that Bax induces the MPT, a critical event in the loss of cell viability. In addition to the cell death, the MPT mediates other typical manifestations of apoptosis in this model, namely release of cytochrome c, caspase activation with PARP cleavage, and DNA fragmentation.

OBJECTIVE

ras oncogenic activation has long been demonstrated in thyroid carcinomas of follicular cell derivation, but no consistent relationship has been shown between mutations and clinicopathologic features.

METHODS

We analyzed H-, K-, and N-ras mutations by polymerase chain reaction-single-strand conformational polymorphism followed by DNA sequencing in 125 thyroid carcinoma specimens from 107 patients, to include tumors covering the entire spectrum of thyroid tumor differentiation.

RESULTS

Mutations were identified in four (8.2%) of 49 well-differentiated carcinomas (WDCs; two [6.7%] of 30 of the tumors were papillary carcinomas, two [10.5%] of 19 of them were follicular carcinomas), in 16 (55.2%) of 29 poorly differentiated carcinomas (PDCs), and in 15 (51.7%) of 29 undifferentiated carcinomas, with a significant association between ras mutation and poorly or undifferentiated tumors (P <.001). Twenty-six (74.3%) of 35 patients with ras-mutated tumors died as a result of disease as opposed to 23 (31.9%) of 72 patients with tumors lacking the mutations. Among patients with differentiated thyroid carcinomas (WDC and PDC), 11 (55.0%) of 20 patients with mutated tumors died as a result of disease as opposed to nine (15.5%) of 58 patients with wild-type ras tumors, and the correlation was independent of tumor differentiation and stage (P =.016). K-ras codon 13 mutations (all with G-A nucleotide transitions resulting in Gly>Asp substitution) and single activating mutations in any of the ras genes were also independent predictors of poor survival in differentiated thyroid carcinomas (P =.027 and P =.007, respectively).

CONCLUSIONS

These findings demonstrate that ras mutations are a marker for aggressive cancer behavior and indicate a possible role of ras genotyping to identify thyroid carcinoma subsets associated with poor prognosis.

Crystallographic studies of neuraminidase-sialic acid complexes indicate that sialic acid is distorted on binding the enzyme. Three arginine residues on the enzyme interact with the carboxylate group of the sugar which is observed to be equatorial to the saccharide ring as a consequence of its distorted geometry. The glycosidic oxygen is positioned within hydrogen-bonding distance of Asp-151, implicating this residue in catalysis.

The Escherichia coli heat shock proteins DnaK and DnaJ function cooperatively as molecular chaperones. Central to their biochemical functions is the ability of DnaJ to interact with DnaK and to stimulate its ATPase activity. Here, we report the genetic isolation of dnaJ12, which has a nonsense mutation at codon 109, yet was able to support lambda growth at 30 degrees C. The 12-kDa DnaJ12 protein was purified to homogeneity and shown to be active in an in vitro lambda-DNA replication system and to be capable of stimulating DnaK's ATPase activity, specifically at the step of ATP hydrolysis. The previously well studied and characterized dnaJ259 mutation was also cloned and sequenced, revealing a single His->>Gln amino acid change at codon 33. The purified DnaJ259 protein was inactive in an in vitro lambda-DNA replication system and was unable to stimulate DnaK's ATPase activity. Consistent with this, an NH2-terminal deletion of the first 34 amino acids or an Asp insertion at residue 35 of DnaJ resulted in a protein that completely lacked DnaJ activity. Collectively, these results demonstrate that the highly conserved NH2-terminal region of DnaJ, the so-called J region, is necessary and sufficient for stimulating both DnaK's ATPase activity and lambda-DNA replication. These results may be applicable to other eukaryotic proteins that contain this conserved J domain as proteins that interact and stimulate the hydrolysis of ATP by their cognate HSP70 proteins.

Amino acid sequences have been compared for thermophilic and mesophilic molecules of ferredoxin, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase. It is shown that Gly, Ser, Ser, Lys, and Asp in mesophiles are generally substituted by Ala, Ala, Thr, Arg, and Glu, respectively, in thermophiles. These exchanges suggest that thermal stability can be achieved by the addition of many small changes throughout the molecule without significant change in the backbone conformation. Their overall effect is primarily to increase internal and decrease external hydrophobicity as well as to favor helix stabilizing residues in helices. These substitutions minimize interruption of function or internal residue packing arrangements. Although the analysis has been confined to the above-mentioned molecules, the observed stabilizing principles may be more generally applicable.

The ATE1-encoded Arg-transferase mediates conjugation of Arg to N-terminal Asp, Glu, and Cys of certain eukaryotic proteins, yielding N-terminal Arg that can act as a degradation signal for the ubiquitin-dependent N-end rule pathway. We have previously shown that mouse ATE1-/- embryos die with defects in heart development and angiogenesis. Here, we report that the ATE1 Arg-transferase mediates the in vivo degradation of RGS4 and RGS5, which are negative regulators of specific G proteins whose functions include cardiac growth and angiogenesis. The proteolysis of these regulators of G protein signaling (RGS) proteins was perturbed either by hypoxia or in cells lacking ubiquitin ligases UBR1 and/or UBR2. Mutant RGS proteins in which the conserved Cys-2 residue could not become N-terminal were long-lived in vivo. We propose a model in which the sequential modifications of RGS4, RGS5, and RGS16 (N-terminal exposure of their Cys-2, its oxidation, and subsequent arginylation) act as a licensing mechanism in response to extracellular and intracellular signals before the targeting for proteolysis by UBR1 and UBR2. We also show that ATE1-/- embryos are impaired in the activation of extracellular signal-regulated kinase mitogen-activated protein kinases and in the expression of G protein-induced downstream effectors such as Jun, cyclin D1, and beta-myosin heavy chain. These results establish RGS4 and RGS5 as in vivo substrates of the mammalian N-end rule pathway and also suggest that the O2-ATE1-UBR1/UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system.

Scoring functions are widely applied to the evaluation of protein-ligand binding in structure-based drug design. We have conducted a comparative assessment of 16 popular scoring functions implemented in main-stream commercial software or released by academic research groups. A set of 195 diverse protein-ligand complexes with high-resolution crystal structures and reliable binding constants were selected through a systematic nonredundant sampling of the PDBbind database and used as the primary test set in our study. All scoring functions were evaluated in three aspects, that is, "docking power", "ranking power", and "scoring power", and all evaluations were independent from the context of molecular docking or virtual screening. As for "docking power", six scoring functions, including GOLD::ASP, DS::PLP1, DrugScore(PDB), GlideScore-SP, DS::LigScore, and GOLD::ChemScore, achieved success rates over 70% when the acceptance cutoff was root-mean-square deviation < 2.0 A. Combining these scoring functions into consensus scoring schemes improved the success rates to 80% or even higher. As for "ranking power" and "scoring power", the top four scoring functions on the primary test set were X-Score, DrugScore(CSD), DS::PLP, and SYBYL::ChemScore. They were able to correctly rank the protein-ligand complexes containing the same type of protein with success rates around 50%. Correlation coefficients between the experimental binding constants and the binding scores computed by these scoring functions ranged from 0.545 to 0.644. Besides the primary test set, each scoring function was also tested on four additional test sets, each consisting of a certain number of protein-ligand complexes containing one particular type of protein. Our study serves as an updated benchmark for evaluating the general performance of today's scoring functions. Our results indicate that no single scoring function consistently outperforms others in all three aspects. Thus, it is important in practice to choose the appropriate scoring functions for different purposes.

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.

The cytoskeletal protein non-erythroid alpha-spectrin is well documented as an endogenous calpain substrate, especially under pathophysiological conditions. In cell necrosis (e.g. maitotoxin-treated neuroblastoma SH-SY5Y cells), alpha-spectrin breakdown products (SBDPs) of 150 kDa and 145 kDa were produced by cellular calpains. In contrast, in neuronal cells undergoing apoptosis (cerebellar granule neurons subjected to low potassium and SH-SY5Y cells treated with staurosporine), an additional SBDP of 120 kDa was also observed. The formation of the 120 kDa SBDP was insensitive to calpain inhibitors but was completely blocked by an interleukin 1 beta-converting-enzyme (ICE)-like protease inhibitor, Z-Asp-CH2OC(O)-2,6-dichlorobenzene. Autolytic activation of both calpain and the ICE homologue CPP32 was also observed in apoptotic cells. alpha-Spectrin can also be cleaved in vitro by purified calpains to produce the SBDP doublet of 150/145 kDa and by ICE and ICE homologues [ICH-1, ICH-2 and CPP32(beta)] to produce a 150 kDa SBDP. In addition, CPP32 and ICE also produced a 120 kDa SBDP. Furthermore inhibition of either ICE-like protease(s) or calpain protects both granule neurons and SH-SY5Y cells against apoptosis. Our results suggest that both protease families participate in the expression of neuronal apoptosis.

The three-dimensional structure of wild-type CheY from Escherichia coli has been refined by stereochemically restrained least squares minimization to a crystallographic R-factor of 15.1% at 1.7-A resolution. The structure contains 1165 atoms, including all atoms of the protein, 147 water molecules, and three sulfate ions. The final model has root mean square deviations of 0.018 and 0.049 A from idealized bond lengths and angle distances, respectively. Seven amino acid side chains have been modeled in dual conformations. CheY folds as a compact (beta/alpha)5 globular protein, with the phosphorylation region contained in a cavity on one face of the molecule. This active site area is bordered by the carboxyl termini of the three central beta-strands, by alpha 1, and by the loop connecting beta 5 to alpha 5. The Lys-109 side chain of this loop extends into the active site by virtue of its cis peptide bond conformation preceding Pro-110. The epsilon-amino group of Lys-109 is in close bonding contact with the carboxyl group of Asp-57, the residue that is phosphorylated in the activation process of CheY. The details of the hydrogen bonding network in the phosphorylation region indicate that structural rearrangements must accompany the phosphorylation of Asp-57.

A cDNA clone encoding the high affinity Ca2+-binding protein (HACBP) of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 418 amino acids, but a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that a 17-residue NH2-terminal signal sequence was removed during synthesis. This was confirmed by studies of in vitro translation of mRNA encoding the protein. Structural predictions did not reveal any potential transmembrane segments in the protein. The COOH-terminal sequence of the high affinity Ca2+-binding protein, Lys-Asp-Glu-Leu, is the same as that proposed to be an endoplasmic reticulum retention signal (Munro, S., and Pelham, H. R. B. (1987) Cell 48, 899-907). All of these characteristics suggest that the protein is localized in the lumen of the sarcoplasmic reticulum. The mature protein of Mr 46,567 contains 109 acidic and 52 basic amino acids. Structural predictions suggest that the first half of the molecule forms a globular domain of 8 anti-parallel beta-strands with a helix-turn-helix motif at the extreme NH2 terminus. The next one-third of the sequence is proline-rich. This segment can be subdivided into a charged region which contains a 17-amino acid repeat, followed by a proline, serine, and threonine-rich segment extending from Pro-246 to Thr-316. Thirty-seven acidic residues are clustered within 56 amino acids at the COOH terminus of the protein. Although the protein binds 1 mol of Ca2+/mol with high affinity, no "EF-hand" consensus sequence was observed in the protein. The acidic COOH terminus, however, could account for the low affinity, high capacity Ca2+ binding observed in the protein. In agreement with other involved laboratories, we have chosen the name calreticulin for the protein.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally regulates hepatic low-density lipoprotein receptors (LDLRs) by binding to LDLRs on the cell surface, leading to their degradation. The binding site of PCSK9 has been localized to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. Here, we describe the crystal structure of a complex between PCSK9 and the EGF-A domain of the LDLR. The binding site for the LDLR EGF-A domain resides on the surface of PCSK9's subtilisin-like catalytic domain containing Asp-374, a residue for which a gain-of-function mutation (Asp-374-Tyr) increases the affinity of PCSK9 toward LDLR and increases plasma LDL-cholesterol (LDL-C) levels in humans. The binding surface on PCSK9 is distant from its catalytic site, and the EGF-A domain makes no contact with either the C-terminal domain or the prodomain. Point mutations in PCSK9 that altered key residues contributing to EGF-A binding (Arg-194 and Phe-379) greatly diminished binding to the LDLR's extracellular domain. The structure of PCSK9 in complex with the LDLR EGF-A domain defines potential therapeutic target sites for blocking agents that could interfere with this interaction in vivo, thereby increasing LDLR function and reducing plasma LDL-C levels.

DNA damage induced by the cancer chemotherapeutic drug etoposide triggers the onset of a series of intracellular events characteristic of apoptosis. Among the early changes observed is the release of cytochrome c from mitochondria, although the mechanism responsible for this effect is unclear. We demonstrate here a role for caspase-2 in etoposide-induced cytochrome c release. In particular, Jurkat T-lymphocytes treated with an irreversible caspase-2 inhibitor, benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VDVAD-fmk), or stably transfected with pro-caspase-2 antisense (Casp-2/AS) are refractory to cytochrome c release stimulated by etoposide. Experiments performed using a reconstituted cell-free system indicate that etoposide-induced cytochrome c release by way of caspase-2 occurs independently of cytosolic factors, suggesting that the nuclear pool of pro-caspase-2 is critical to this process. Apart from inhibiting cytochrome c release, undermining caspase-2 activity results in an attenuation of downstream events, such as pro-caspase-9 and -3 activation, phosphatidylserine exposure on the plasma membrane, and DNA fragmentation. Taken together, our data indicate that caspase-2 provides an important link between etoposide-induced DNA damage and the engagement of the mitochondrial apoptotic pathway.

In plants, the cytokinin (CK) phytohormones regulate numerous biological processes, including responses to environmental stresses, via a complex network of CK signaling. By an unknown mechanism, stress signals are perceived and transmitted through the His-Asp phosphorelay, an important component of the CK signal transduction pathway, triggering CK-responsive genes. Because of the intensive crosstalk between CKs and abscisic acid (ABA), modulation of CK levels and their signal transduction affects both ABA-dependent and ABA-independent pathways, enabling plant adaptation to adverse conditions. This review presents our current understanding of the functions of CKs and CK signaling in the regulation of plant adaptation to stress. Biotechnological strategies based on the modulation of CK levels have been examined with the aim of stabilizing agriculture yields.

Protein arginine methylation has been implicated in signal transduction, nuclear transport and transcription regulation. Protein arginine methyltransferases (PRMTs) mediate the AdoMet-dependent methylation of many proteins, including many RNA binding proteins involved in various aspects of RNA processing and/or transport. Here we describe the crystal structure of the rat PRMT3 catalytic core in complex with reaction product AdoHcy, determined at 2.0 A resolution. The results reveal a two-domain structure: an AdoMet-binding domain and a barrel-like domain. The AdoMet-binding domain is a compact version of the consensus AdoMet-dependent methyltransferase fold. The active site is situated in a cone-shaped pocket between the two domains. The residues that make up the active site are conserved across the PRMT family, consisting of a double-E loop containing two invariant Glu and one His-Asp proton-relay system. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the PRMT family. In addition, crystal packing and solution behavior suggest dimer formation of the PRMT3 core.

Glioblastoma multiforme, the most malignant astroglial-derived tumor, grows as an adherent mass and locally invades normal brain. An examination of adult cerebral glioblastoma biopsy material for the expression of adhesive proteins that might potentiate adhesion and invasion demonstrated tumor cell-associated vitronectin (5/5). In contrast, vitronectin was not detected associated with glial cells in low grade astroglial tumors (0/4), reactive astrogliosis (0/4), or in normal adult cortex and cerebral white matter (0/5). Also, a wide variety of other adhesive ligands were absent from the glioblastoma tumor parenchyma. The alpha v beta 3 integrin was the only vitronectin receptor identified in glioblastoma tumors in situ, and was also not expressed on low grade astroglial-derived tumors, reactive astrogliosis, or on glia or neurons in normal adult cortex and cerebral white matter. In a cell attachment assay, cultured glioblastoma cells attached to the parenchyma of glioblastoma tumor cryostat sections at the sites of vitronectin expression, but failed to attach to normal brain. This adhesion was inhibited by antibodies directed against vitronectin, the alpha v beta 3 integrin, and with an Arg-Gly-Asp-containing peptide. These data provide evidence for a cell adhesion mechanism in glioblastoma tumors that might potentiate glioblastoma cell invasion of normal brain.

The cell surface receptor for fibronectin is a heterodimeric membrane protein that recognizes an Arg-Gly-Asp sequence in fibronectin and that requires cations such as Mg2+ or Ca2+ for binding to fibronectin. The divalent cation requirements of this receptor were analyzed by measuring attachment of receptor liposomes to ligand-coated surfaces in the presence of different cations. The most striking effect observed was produced by Mn2+, which increased the binding of the receptor liposomes to fibronectin 2-3-fold over their binding in buffers containing Ca2+ and Mg2+. The binding activities of two related adhesion receptors, the vitronectin receptor and platelet GP IIb-IIIa, were supported but not enhanced by Mn2+. Two observations suggest that Mn2+ can compete with Ca2+ for the same cation-binding sites of the receptor. First, Mn2+ could still enhance fibronectin receptor binding activity even in the presence of 10-fold higher concentrations of Ca2+ or Mg2+. Second, Mn2+ inhibited the binding of radioactive Ca2+ to the alpha subunit of the receptor. The increased fibronectin receptor activity in the presence of Mn2+ appeared to be due to an increase in the affinity of the receptor for the Arg-Gly-Asp sequence because a 110-kDa cell attachment fragment and a synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited liposome binding more effectively in the presence of Mn2+ than in the presence of Ca2+/Mg2+. The affinity for the peptide was affected more than the affinity for the fragment, indicating that Mn2+ also induces a change in receptor specificity. Increased receptor binding in the presence of Mn2+ was also apparent in affinity chromatography of the fibronectin receptor on the 110-kDa fibronectin fragment; Mn2+ improved the yield of the receptor 4-fold. Mn2+ similarly increased the number of receptor-fibronectin complexes in preparations analyzed by electron microscopy. These results show that exogenous influences can modulate the affinity and specificity with which the fibronectin receptor binds to its ligands.

During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.

Protein tyrosine kinases activate the STAT (signal transducer and activator of transcription) signaling pathway, which can play essential roles in cell differentiation, cell cycle control, and development. However, the potential role of the STAT signaling pathway in the induction of apoptosis remains unexplored. Here we show that gamma interferon (IFN-gamma) activated STAT1 and induced apoptosis in both A431 and HeLa cells, whereas epidermal growth factor (EGF) activated STAT proteins and induced apoptosis in A431 but not in HeLa cells. EGF receptor autophosphorylation and mitogen-activated protein kinase activation in response to EGF were similar in both cell lines. The breast cancer cell line MDA-MB-468 exhibited a similar response to A431 cells, i.e., STAT activation and apoptosis correlatively resulted from EGF or IFN-gamma treatment. In addition, in a mutant A431 cell line in which STAT activation was abolished, no apoptosis was induced by either EGF or IFN-gamma. We further demonstrated that both EGF and IFN-gamma induced caspase 1 (interleukin-1beta converting enzyme [ICE]) gene expression in a STAT-dependent manner. IFN-gamma was unable to induce ICE gene expression and apoptosis in either JAK1-deficient HeLa cells (E2A4) or STAT1-deficient cells (U3A). However, ICE gene expression and apoptosis were induced by IFN-gamma in U3A cells into which STAT1 had been reintroduced. Moreover, both EGF-induced apoptosis and IFN-gamma-induced apoptosis were effectively blocked by Z-Val-Ala-Asp-fluoromethylketone (ZVAD) in all the cells tested, and studies from ICE-deficient cells indicated that ICE gene expression was necessary for IFN-gamma-induced apoptosis. We conclude that activation of the STAT signaling pathway can induce apoptosis through the induction of ICE gene expression.

When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a half-life of approximately 10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-Arg-Gly-Asp-Ser-coated microbeads (approximately 10 microbeads/cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene-transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis.

BACKGROUND

The Laparoscopic approach has been applied to colorectal surgery for many years; however, there are only a few reports on laparoscopic low and ultralow anterior resection with construction of coloanal anastomosis. This study compares open versus laparoscopic low and ultralow anterior resections, assesses the feasibility and efficacy of the laparoscopic approach of total mesorectal excision (TME) with anal sphincter preservation (ASP), and analyzes the short-term results of patients with low rectal cancer.

METHODS

We analyzed our experience via a prospective, randomized control trail. From June 2001 to September 2002, 171 patients with low rectal cancer underwent TME with ASP, 82 by the laparoscopic procedure and 89 by the open technique. The lowest margin of tumors was below peritoneal reflection and 1.5-8 cm above the dentate line (1.5-4.9 cm in 104 cases and 5-8 cm in 67 cases). The grouping was randomized.

RESULTS

Results of operation, postoperative recovery, and short-term oncological follow-up were compared between 82 laparoscopic procedures and 89 controls who underwent open surgery during the same period. In the laparoscopic group, 30 patients in whom low anterior resection was performed had the anastomosis below peritoneal reflection and more than 2 cm above the dentate line, 27 patients in whom ultralow anterior resection was performed had anastomotic height within 2 cm of the dentate line, and 25 patients in whom coloanal anastomosis was performed had the anastomosis at or below the dentate line. In the open group, the numbers were 35, 27, and 27, respectively. There was no statistical difference in operation time, administration of parenteral analgesics, start of food intake, and mortality rate between the two groups. However, blood loss was less, bowel function recovered earlier, and hospitalization time was shorter in the laparoscopic group.

CONCLUSIONS

Totally laparoscopic TME with ASP is feasible, and it is a minimally invasive technique with the benefits of much less blood loss during operation, earlier return of bowel function, and shorter hospitalization.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.

Purified integrase protein (IN) can nick linear viral DNA at a specific site near the ends and integrate nicked viral DNA into target DNA. We have made a series of 43 site-directed point mutants of human immunodeficiency virus type 2 IN and assayed purified mutant proteins for the following activities: site-specific cleavage of viral DNA (donor cut), integration (strand transfer), and disintegration. In general, the different activities were similarly affected by the mutations. We found three mutations that (almost) totally abolished IN function: Asp-64->>Val, Asp-116->>Ile, and Glu-152->>Leu, whereas 25 mutations did not affect IN function. A few mutations affected the different activities differentially. Near the amino terminus a zinc finger-like sequence motif His-Xaa3-His-Xaa20-30-Cys-Xaa2-Cys is present in all retroviral IN proteins. Two mutations in this region (His-12->>Leu and Cys-40->>Ser) strongly inhibited donor cut but had less effect on strand transfer. The central region of IN is most highly conserved between retroviral INs. Three mutants in this region (Asn-117->>Ile, Asn-120->>Leu, and Lys-159->>Val) were inhibited in strand transfer but were inhibited less strongly in donor cut. Mutation of Asn-120 (to glycine, leucine, or glutamate) resulted in changes in integration-site preference, suggesting that Asn-120 is involved in interactions with target DNA. We did not find a mutant in which one activity was lost and the others were unaffected, supporting the notion that IN has only one active site for the catalysis of donor cut and strand transfer.