Defining MAP3 kinases required for MDA-MB-231 cell tumor growth and metastasis.

Analysis of patient tumors suggests that multiple MAP3 kinases (MAP3Ks) are critical for growth and metastasis of cancer cells. MAP3Ks selectively control the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), p38 and ERK5 in response to receptor tyrosine kinases and GTPases. We used MDA-MB-231 cells because of their ability to metastasize from the breast fat pad to distant lymph nodes for an orthotopic xenograft model to screen the function of seven MAP3Ks in controlling tumor growth and metastasis. Stable short hairpin RNA (shRNA) knockdown was used to inhibit the expression of each of the seven MAP3Ks, which were selected for their differential regulation of the MAPK network. The screen identified two MAP3Ks, MEKK2 and MLK3, whose shRNA knockdown caused significant inhibition of both tumor growth and metastasis. Neither MEKK2 nor MLK3 have been previously shown to regulate tumor growth and metastasis in vivo. These results demonstrated that MAP3Ks, which differentially activate JNK, p38 and ERK5, are necessary for xenograft tumor growth and metastasis of MDA-MB-231 tumors. The requirement for MAP3Ks signaling through multiple MAPK pathways explains why several members of the MAPK network are activated in cancer. MEKK2 was required for epidermal growth factor receptor and Her2/Neu activation of ERK5, with ERK5 being required for metastasis. Loss of MLK3 expression increased mitotic infidelity and apoptosis in vitro. Knockdown of MEKK2 and MLK3 resulted in increased apoptosis in orthotopic xenografts relative to control tumors in mice, inhibiting both tumor growth and metastasis; MEKK2 and MLK3 represent untargeted kinases in tumor biology for potential therapeutic development.

Duplex ultrasound assessment of femoral venous flow during laparoscopic and open gastric bypass.

BACKGROUND: Pneumoperitoneum (PP) and the reverse Trendelenburg (RT) position have been shown to decrease femoral blood flow, resulting in venous stasis. However the effects of PP and RT on femoral venous flow have not been evaluated in morbidly obese patients undergoing laparoscopic gastric bypass (GBP). We analyzed the effects of PP and RT on peak systolic velocity and the cross-sectional area of the femoral vein during laparoscopic and open GBP. We further examined the efficacy of intermittent sequential compression devices in reversing the reduction of femoral peak systolic velocity. METHODS: Thirty patients with a body mass index (BMI) of 40-60 were randomly allocated to under go either laparoscopic (n = 14) or open (n = 16) GBP. A duplex ultrasound examination of the femoral vein was performed at baseline, during PP and combined PP and RT in the laparoscopic group, and at baseline and during RT in the open group. The ultrasound exam was performed first without the use of sequential compression devices and then with the sequential compression devices inflated to 45 mmHg. RESULTS: The two groups were similar in age, sex, BMI, and calf and thigh circumferences. During laparoscopic GBP, PP resulted in a 43% decrease in peak systolic velocity and a 52% increase in the cross-sectional area of the femoral vein; the combination of PP and RT decreased peak systolic velocity to 57% of baseline and increased the femoral cross-sectional area to 121% of baseline. During laparoscopic GBP, the use of sequential compression devices during PP and RT partially reversed the reduction of femoral peak systolic velocity, but femoral peak systolic velocity was still lower than baseline by 38%. During open GBP, RT resulted in a 38% reduction in peak systolic velocity and a 69% increase in the cross-sectional area of the femoral vein; the use of sequential compression devices during RT partially reversed these changes by increasing femoral peak systolic velocity by 26%; however, it was still lower than baseline by 22%. CONCLUSIONS: Pneumoperitoneum and reverse Trendelenburg position during laparoscopic and open GBP are independent factors for the development of venous stasis. Combining the reverse Trendelenburg position with pneumoperitoneum during laparoscopic GBP further reduces femoral peak systolic velocity and hence increases venous stasis. The use of sequential compression devices was partially effective in reversing the reduction of femoral peak systolic velocity, but it did not return femoral peak systolic velocity to baseline levels.

Ultrasonographic evaluation of diaphragmatic motion.

OBJECTIVE: To evaluate the technical feasibility and utility of ultrasonography in the study of diaphragmatic motion at our institution. METHODS: The study consisted of 2 parts. For part I, in 23 volunteers we performed 23 studies on 46 hemidiaphragms with excursions documented on M-mode ultrasonography For part II, in 22 patients we performed 52 studies in 102 hemidiaphragms. In 50 studies both hemidiaphragms were studied, and in another 2 studies only 1 hemidiaphragm was studied. Patients' ages ranged from birth to 66 years (mean, 23 years). There were 16 male and 6 female patients. Indications for the study were (1) suggestion of paralysis of the diaphragm (n = 22); (2) if the diaphragm was already known to be paralyzed, for evaluation of response to phrenic nerve or pacer stimulation (n = 9); and (3) follow-up of previous findings (n = 21). Patients were examined in the supine position in the longitudinal semicoronal plane from a subcostal or low intercostal approach. Motion was documented with real-time ultrasonography and measured with M-mode ultrasonography. RESULTS: Of the 102 clinical hemidiaphragms studied, findings included normal motion (n = 42), decreased motion (n = 22), no motion (n = 6), paradoxical motion (n = 10), positive pacer response (n = 13), negative pacer response (n = 2), positive phrenic stimulation (n = 6), and negative phrenic stimulation (n = 1). There were no failures of visualization. CONCLUSIONS: Ultrasonography proved feasible and useful in evaluating diaphragmatic motion. In our practice it has replaced fluoroscopy. Ultrasonography has advantages over traditional fluoroscopy, including portability, lack of ionizing radiation, visualization of structures of the thoracic bases and upper abdomen, and the ability to quantify diaphragmatic motion.

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus.

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

Comparison of US utilization and technical costs before and after establishment of 24-hour in-house coverage for US examinations.

PURPOSE: To compare data regarding the cost and number of ultrasonographic (US) examinations performed for 6 months, before and after institution of 24-hour in-house sonographer coverage. MATERIALS AND METHODS: Data for a 6-month period during which US services were provided by a sonographer on call from 11 PM to 7 AM were compared with data for a 6-month period during which a sonographer was in house during this shift. RESULTS: With 11 PM to 7 AM on-call coverage, the sonographers performed 147 examinations in a 6-month period, an average of 0.81 examination per shift. After institution of in-house coverage for this shift, 792 US examinations were performed in 6 months, an average of 4.3 examinations per shift. The cost for 11 PM to 7 AM in-house sonographer coverage for 6 months was approximately $16,000 more than that for on-call coverage. This cost would be offset by revenues from one additional examination per night. The cost per examination for the 11 PM to 7 AM shift decreased from $124.70 to $43.33. CONCLUSION: At the authors' institution, 24-hour in-house sonographer coverage resulted in additional cost, which was offset by revenues from additional examinations. There was nearly a fivefold increase in the number of US examinations performed per shift. These examinations were performed more expediently, enabling more rapid patient triage.

Mutations in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage.

The mechanisms responsible for macrolide resistance in Streptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A), erm(A), erm(B), erm(C), erm(TR), msr(A), mph(A), mph(B), mph(C), ere(A), ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia coli numbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids ((63)KPWRQKGTGRAR(74)) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.

Spinal sonography and magnetic resonance imaging in patients with repaired myelomeningocele: comparison of modalities.

The goals of this study were to evaluate the feasibility of using ultrasonography of the spine in the follow-up evaluation of patients with repaired myelomeningocele at birth and to compare sonography with the accepted modality of magnetic resonance imaging. Over a period of 4 years we performed 165 sonographic studies in 101 patients; 107 sonographic studies had MR imaging results for comparison. We collected our data prospectively. The quality of the sonograms was good in 110 of 129 studies, acceptable in 17 of 129, and poor in two of 129. The sonographic examinations failed in 33 of 165 studies (20%). Concordant information was obtained between ultrasonography and magnetic resonance imaging in the following percentage of studies: level of the distal end of the cord in 82%, position of the cord in the canal in 59%, presence of hydromyelia in 63%, cord duplication in 96%, adhesions in 16%, intradural mass in 37%, cord measurements in 85%, and dural sac measurements in 83%. At the lumbosacral level, we saw no cord pulsation in 57% of the studies in patients with cord adhesions and in 20% of those without adhesions. At the lower thoracic level, we saw no pulsation in 35% of the studies in patients with cord adhesions and in 7% of those without adhesions. Postoperative studies of cord release surgery in eight patients showed varied findings. We conclude that in those patients who have a spinal defect or interlaminar space allowing proper visualization of the lumbosacral spinal canal, ultrasound can provide fairly similar information to that obtained with magnetic resonance imaging of that area with no need for sedation and at a reduced cost. Ultrasonography seems more sensitive than magnetic resonance imaging in the detection of cord adhesions, which is particularly relevant in the diagnosis of tethering.

mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae.

Recently, it was shown that a significant number of erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains contain a determinant that mediates resistance via a putative efflux pump. The gene encoding the erythromycin-resistant determinant was cloned and sequenced from three strains of S. pneumoniae bearing the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible). The DNA sequences of mefE were nearly identical, with only 2-nucleotide differences between genes from any two strains. When the mefE sequences were compared to the mefA sequence from S. pyogenes, the two genes were found to be closely related (90% identity). Strains of S. pneumoniae were constructed to confirm that mefE is necessary to confer erythromycin resistance and to explore the substrate specificity of the pump; no substrates other than 14- and 15-membered macrolides were identified.

Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes.

Several streptococcal strains had an uncharacterized mechanism of macrolide resistance that differed from those that had been reported previously in the literature. This novel mechanism conveyed resistance to 14- and 15-membered macrolides, but not to 16-membered macrolides, lincosamides or analogues of streptogramin B. The gene encoding this phenotype was cloned by standard methods from total genomic digests of Streptococcus pyogenes 02C1064 as a 4.7 kb heterologous insert into the low-copy vector, pACYC177, and expressed in several Escherichia coli K-12 strains. The location of the macrolide-resistance determinant was established by functional analysis of deletion derivatives and sequencing. A search for homologues in the genetic databases confirmed that the gene is a novel one with homology to membrane-associated pump proteins. The macrolide-resistance coding sequence was subcloned into a pET23a vector and expressed from the inducible T7 promoter on the plasmid in E. coli BL21(DE3). Physiological studies of the cloned determinant, which has been named mefA for macrolide efflux, provide evidence for its mechanism of action in host bacteria. E.coli strains containing the cloned determinant maintain lower levels of intracellular erythromycin when this compound is added to the external medium than isogenic clones without mefA. Furthermore, intracellular accumulation of [14C]-erythromycin in the original S. pyogenes strain was always lower than that observed in erythromycin-sensitive strains. This is consistent with a hypothesis that the gene encodes a novel antiporter function which pumps erythromycin out of the cell. The gene appears to be widely distributed in S. pyogenes strains, as demonstrated by primer-specific synthesis using the polymerase chain reaction.

Accuracy and precision of in vitro volumetric measurements by three-dimensional sonography.

RATIONALE AND OBJECTIVES: A radiologist often wishes to measure organ volume or monitor changes in internal lesion volume during treatment. If this can be determined via three-dimensional ultrasound, the relative simplicity of the procedure and the decreased cost and known risks to the patient would make this method an attractive alternative to other modalities. METHODS: Three-dimensional ultrasound scans were made of six phantoms: four nonechogenic spheres, one echogenic sphere, and one echogenic, irregularly shaped phantom. A total of 22 volume scans were produced. Volume estimations were made using data from cross-sectional areas and from linear measurements. In all, 193 volume estimations were made. These results were compared with known volumes and with volume estimates from computed tomography scans. RESULTS: Three-dimensional ultrasound detected size differences of 10% with 95% certainty. CONCLUSIONS: The accuracy and precision of volume estimates via three-dimensional ultrasound is at least as good as those obtained via conventional ultrasound.

Inhibition of interleukin-1 beta production by SKF86002: evidence of two sites of in vitro activity and of a time and system dependence.

Cytokine-suppressing anti-inflammatory drugs (CSAIDs) are reported to inhibit production of proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) by affecting a stress-induced kinase. To gain a better understanding of the selectivity and cellular dynamics of this type of inhibitor, we studied in vitro the prototype member of this class of agents, SKF86002. Lipopolysaccharide (LPS)-activated human monocytes treated with SKF86002 produced less proIL-1 beta but normal amounts of the noncytokine lysozyme. Two-dimensional gel analysis indicated that only eight polypeptides produced by monocytes were decreased by SKF86002. Inhibition of IL-1 beta production was achieved by affecting two separate steps in this cytokine's biogenesis. First, SKF86002 lowered proIL-1 beta synthesis. By pulse-chase analysis, this effect was localized to a posttranscriptional site of action; maximal inhibition was observed when SKF86002 was added at the time of cytokine translation. Exposure of monocytes to SKF86002 for > 2 hr led to a loss of IL-1 beta inhibitory activity, suggesting that these cells adapted to this agent. Moreover, LPS-activated monocytes that were pretreated with granulocyte-macrophage colony-stimulating factor were less sensitive to the proIL-1 beta inhibitory effect of SKF86002, and production of proIL-1 beta by cytokine-stimulated human fibroblasts was impaired only modestly by the CSAID. A second effect of SKF86002 was to inhibit release of IL-1 beta into the medium in response to high concentrations of LPS; this effect is observed only with freshly isolated human monocytes as other IL-1 beta-producing cells do not release significant cytokine in response to LPS. The ability of SKF86002 to inhibit this posttranslational mechanism was mimicked by lysosomotrophic agents such as chloroquine, quinacrine, and methylamine. In contrast, chloroquine, and quinacrine were not effective inhibitors of monocyte proIL-1 beta translation. Thus, SKF86002 inhibits IL-1 beta production by affecting at least two distinct steps in the biosynthesis of this cytokine. Manifestation of these two effects, however, is dependent on the length of time for which cells are exposed to this agent and the nature of the cytokine-producing cellular system.

Proximal fetal cerebral ventricle: description of US technique and initial results.

PURPOSE: To establish adequacy and ease of visualization of the proximal ventricle, normal range of measurements of the proximal ventricle, and distance of the proximal choroid plexus from the lateral ventricular wall. MATERIALS AND METHODS: With use of an angled technique, ultrasound (US) evaluation of the proximal fetal ventricle was attempted in 439 fetuses during routine obstetric US examination. Ease of examination, additional time required, mean measurements, and standard deviation (SD) were calculated. RESULTS: Visualization and measurement of the proximal ventricle were performed without difficulty in 77% of cases and with difficulty in 19%, and were impossible in 4%. Average additional time required was 4.2 minutes. The upper limit of normal for the midportion of the proximal ventricle was 8 mm (mean + 2.5 SD). In no normal pregnancy was the proximal ventricle separated from the choroid plexus by greater than 3 mm. CONCLUSION: Visualization and measurement of the proximal fetal cerebral ventricle can be performed during routine obstetric US examination in little additional time and can be used to detect abnormalities that might otherwise be overlooked because of fetal position.

Ultrasonography: not useful in detecting susceptibility to malignant hyperthermia.

MH is a rare, potentially fatal complication of general anesthesia. Halothane-caffeine contracture testing of a muscle biopsy is the only accepted diagnostic test for MH. A previous report indicated that ultrasonography may aid in diagnosis of MH. Using sonographic examination of the thigh and calf, we evaluated eight patients with proved susceptibility to MH and eight control patients. Two radiologists independently evaluated the sonograms for echogenicity and definition of fascial planes. We detected no consistent and reliable differences between control and MH patients. We conclude that, in our hands, ultrasonography is not useful in differentiating patients with MH from normal persons.

"Spot transfer", elution and comigration with known proteins allows accurate transferral of protein identifications between distinct two-dimensional electrophoretic systems.

We report a simple method, designated "spot transfer", where known proteins are excised and eluted from a two-dimensional (2-D) gel run on one gel system to transfer identification of the proteins to a different 2-D gel system by comigration with a comparable sample. In one experiment, 8 of 16 proteins eluted from an isoelectric focusing (IEF) 2-D gel, of the format described for the Celis human keratinocyte database (Celis, J. E. et al., Electrophoresis 1993, 14, 1091-1198), were found to comigrate with proteins in a human T lymphoma (JURKAT) whole cell lysate run using the Millipore Investigator 2-D system. The method should have general utility in allowing the exchange of protein identifications between investigators and could be used to standardize gel loci used in 2-D gel protein databases.

Three-dimensional sonographic evaluation of developmental dysplasia of the hip: preliminary findings.

PURPOSE: To determine the value of three-dimensional (3D) sonography in the evaluation of developmental dysplasia of the hip. MATERIALS AND METHODS: 3D reconstruction and section analysis were performed on 38 data acquisitions obtained in nine patients with a clinical diagnosis of developmental dysplasia of the hip. Data were obtained mostly in the coronal plane, and section-analysis and 3D volume reconstruction images were generated. RESULTS: Of the 32 image sets obtained in the coronal plane, the technical quality of 27 (84%) section-analysis images and 25 (78%) spatial-revolving images was judged to be satisfactory. CONCLUSION: In addition to permitting global visualization of the hip, 3D sonography offers imaging in the sagittal and craniocaudal projections, something no other modality can offer. 3D sonography can also demonstrate the relationship of the femoral head to the acetabulum and femoral head containment more thoroughly than does conventional sonography.