The anti-angiogenic VEGF isoform VEGF165b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo.

Vascular endothelial growth factor (VEGF) is the principal agent that increases microvascular permeability during physiological and pathological angiogenesis. VEGF is differentially spliced to form two families of isoforms: VEGF(xxx), and VEGF(xxx)b. Whereas VEGF(165) stimulates angiogenesis, VEGF(165)b is anti-angiogenic. To determine the effect of VEGF(165)b on permeability, hydraulic conductivity (L(p)) was measured in individually perfused microvessels in the mesentery of frogs and rats. As with VEGF(165), VEGF(165)b increased L(p) in amphibian (2.4 +/- 0.3-fold) and mammalian (1.9 +/- 0.2-fold) mesenteric microvessels. A dose-response relationship showed that VEGF(165)b (EC(50), 0.65 pm) was approximately 25 times more potent than VEGF(165) (EC(50), 16 pm) in amphibian microvessels. VEGF(165) has been shown to increase permeability through VEGF receptor 2 (VEGF-R2) signalling. However, VEGF(165)b increased L(p) of frog vessels to the same extent in the presence of the VEGF-R2 inhibitor ZM323881, indicating that it does not increase permeability via VEGF-R2 signalling, and was inhibited by the VEGF receptor inhibitor SU5416 at doses that are specific for VEGF receptor 1 (VEGF-R1). VEGF(165)b, in contrast to VEGF(165), did not result in a sustained chronic increase in L(p). These results show that although VEGF(165)b is anti-angiogenic in the mesentery, it does signal in endothelial cells in vivo resulting in a transient, but not sustained, increase in microvascular L(p), probably through VEGF-R1.

Role of endothelial Ca2+ stores in the regulation of hydraulic conductivity of Rana microvessels in vivo.

Vascular permeability is regulated by endothelial cytosolic Ca(2+) concentration ([Ca(2+)](i)). To determine whether vascular permeability is dependent on extracellular Ca(2+) influx or release of Ca(2+) from stores, hydraulic conductivity (L(p)) was measured in single perfused frog mesenteric microvessels in the presence and absence of Ca(2+) influx and store depletion. Prevention of Ca(2+) reuptake into stores by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibition increased L(p) in the absence of extracellular Ca(2+) influx. L(p) was further increased when Ca(2+) influx was restored. Depletion of the Ca(2+) stores with ionomycin and SERCA inhibition increased L(p) in the presence and the absence of extracellular Ca(2+) influx. However, store depletion in itself did not significantly increase L(p) in the absence of active Ca(2+) release from stores into the cytoplasm. There was a significant positive correlation between baseline permeability and the magnitude of the responses to both Ca(2+) store release and Ca(2+) influx, indicating that the Ca(2+) regulating properties of the endothelial cells may regulate the baseline L(p). To investigate the role of Ca(2+) stores in regulation of L(p), the relationship between SERCA inhibition and store release was studied. The magnitude of the L(p) increase during SERCA inhibition significantly and inversely correlated with that during store release by Ca(2+) ionophore, implying that the degree of store depletion regulates the size of the increase on L(p). These data show that microvascular permeability in vivo can be increased by agents that release Ca(2+) from stores in the absence of Ca(2+) influx. They also show that capacitative Ca(2+) entry results in increased L(p) and that the size of the permeability increase can be regulated by the degree of Ca(2+) release.

Community-care waiting list for persons with spinal cord injury.

OBJECTIVES: To disseminate the concept of community care waiting lists for spinal cord injury (SCI) patients with particular reference to carer support for management of neuropathic bladder by a regime of intermittent catheterisation. METHODOLOGY: The surgical waiting list focuses only on operative procedures, and ignores the wider requirements for ensuring satisfactory rehabilitation of people with spinal cord injury in the community. A community-care waiting list for individuals with spinal cord injury should include the following aspects of community care: (1) Home adaptation; (2) Provision of appropriate mobility needs (including wheelchair and cushion); (3) Equipment for comfortable living (including provision of hoist, pressure relieving mattress); (4) Psychological support for spinal cord injury patients and their partners; (5) Nursing home or residential care placement where appropriate; (6) Carer support for global management of complex needs associated with spinal cord injury (eg neuropathic bladder and bowel). RESULTS: Whereas full physical adaptation of the home can wait for some time after discharge, carer support for intermittent catheterisation is required from the first day after discharge from a spinal unit. Lack of such support means that some SCI patients are discharged with long-term indwelling urinary catheters, even though clean intermittent catheterisation is known to be the safest regime for managing the neuropathic bladder. Therefore, the absence of a community care waiting list means that best practice cannot be achieved for some tetraplegic subjects. CONCLUSION: We believe that a community care waiting list for bladder management will help to provide optimum care for neuropathic bladder and, hopefully, reduce the complications related to long-term indwelling catheters in spinal cord injury patients.

Application of an artificial neural network to predict specific class I MHC binding peptide sequences.

Computational methods were used to predict the sequences of peptides that bind to the MHC class I molecule, K(b). The rules for predicting binding sequences, which are limited, are based on preferences for certain amino acids in certain positions of the peptide. It is apparent though, that binding can be influenced by the amino acids in all of the positions of the peptide. An artificial neural network (ANN) has the ability to simultaneously analyze the influence of all of the amino acids of the peptide and thus may improve binding predictions. ANNs were compared to statistically analyzed peptides for their abilities to predict the sequences of K(b) binding peptides. ANN systems were trained on a library of binding and nonbinding peptide sequences from a phage display library. Statistical and ANN methods identified strong binding peptides with preferred amino acids. ANNs detected more subtle binding preferences, enabling them to predict medium binding peptides. The ability to predict class I MHC molecule binding peptides is useful for immunolological therapies involving cytotoxic-T cells.

The internal mammary artery and vein as a recipient site for free-flap breast reconstruction: a report of 110 consecutive cases.

The most common recipient site for free-flap breast reconstruction is the subscapular system. Because of a number of problems we encountered using this recipient site, we became interested in revisiting the internal mammary vessels. This paper reports the use of the internal mammary artery and vein as a recipient site in 110 consecutive cases of breast reconstruction in 87 patients. We also report the technical details of recipient-vessel dissection and vessel size in our series. Complications encountered in the series are discussed, and the advantages of using the internal mammary vessels are enumerated. Our experience of a 99 percent successful flap transfer rate supports the reliability of these recipient vessels in breast reconstructions.

Prevention of preterm birth. Role of daily telephone contact.

OBJECTIVE: To compare the effectiveness of routine management of patients at high risk for preterm delivery to the effectiveness of routine management in combination with daily telephone nursing contact. STUDY DESIGN: The control group, 1 (n = 21), had education and frequent prenatal visits and cervical examinations. The study group, 2 (n = 21), had education, frequent prenatal visits and cervical examination, and daily telephone contact. Group 3 (n = 22) received education but refused to participate. RESULTS: There were no significant differences (SD) between groups in race, smoking, age, multiple gestation, visits, diagnosis of premature labor, mean days gained after diagnosis of premature labor, tocolytic use or bed rest. There was also no SD in preterm birth rate, mode of delivery, number of maternal or neonatal hospital days, mean neonatal weight or gestational age at delivery between groups. While not reducing the overall incidence of preterm birth, this management for all groups resulted in a more advanced gestational age at the time of delivery (mean change = 7.5 weeks, P < .0001) when compared to the patient's first preterm birth. CONCLUSION: This study indicated that daily contact, while providing reassurance and support, did not change the outcome when the study group was compared to women managed similarly but without daily contact.

The latissimus dorsi/scapular bone flap (the "latissimus/bone flap").

During the past 4 years, our trauma and reconstructive service has treated a number of patients with lower extremity trauma involving the loss of both soft tissue and significant segments of tibia. While there are many methods for reconstruction of such defects, we became interested in providing a one-stage reconstruction of both the soft tissues and the missing bone segments. Since our standard flap for lower extremity reconstruction is a latissimus dorsi flap, we became interested in transferring a portion of the lateral border of the scapula along with the latissimus muscle. We dissected 34 cadaver scapulas in order to verify the reliability of the blood supply to the lateral border of the scapula based on the thoracodorsal artery. We then performed 12 "latissimus/bone flaps" from 1988 to 1992. Prior to flap transfer, control of the wound was obtained with surgical debridement and aggressive wound management. The flap usually was performed 5 to 7 days after initial contact with the patient. The muscle was skin grafted. All patients reported are ambulating, with x-ray evidence of bony incorporation of the transferred bone segment into the tibia. We feel that inclusion of the lateral scapula bone with the latissimus dorsi is a useful adjunct in the management of lower extremity trauma.

The alpha-helical rod domain of human lamins A and C contains a chromatin binding site.

We examined regions of human lamins A and C involved in binding to surfaces of mitotic chromosomes. An Escherichia coli expression system was used to produce full-length lamin A and lamin C, and truncated lamins retaining the central alpha-helical rod domain (residues 34-388) but lacking various amounts of the amino-terminal 'head' and carboxy-terminal 'tail' domains. We found that lamin A, lamin C and lamin fragments lacking the head domain and tail sequences distal to residue 431 efficiently assembled into paracrystals and strongly associated with mitotic chromosomes. Furthermore, the lamin rod domain also associated with chromosomes, although efficient chromosome coating required the pH 5-6 conditions needed to assemble the rod into higher order structures. Biochemical assays showed that chromosomes substantially reduced the critical concentration for assembly of lamin polypeptides into pelletable structures. Association of the lamin rod with chromosomes was abolished by pretrypsinization of chromosomes, and was not seen for vimentin (which possesses a similar rod domain). These data demonstrate that the alpha-helical rod of lamins A and C contains a specific chromosome binding site. Hence, the central rod domain of intermediate filament proteins can be involved in interactions with other cellular structures as well as in filament assembly.

CENP-B is a highly conserved mammalian centromere protein with homology to the helix-loop-helix family of proteins.

CENP-B is a centromere associated protein originally identified in human cells as an 80 kDa autoantigen recognized by sera from patients with anti-centromere antibodies (ACA). Recent evidence indicates that CENP-B interacts with centromeric heterochromatin in human chromosomes and may bind to a specific subset of human alphoid satellite DNA. CENP-B has not been unambiguously identified in non-primates and could, in principal, be a primate-specific alphoid DNA binding protein. In this work, a human genomic DNA segment containing the CENP-B gene was isolated and subjected to DNA sequence analysis. In vitro expression identified the site for translation initiation of CENP-B, demonstrating that it is encoded by an intronless open reading frame (ORF) in human DNA. A homologous mouse gene was also isolated and characterized. It was found to possess a high degree of homology with the human gene, containing an intronless ORF coding for a 599 residue polypeptide with 96% sequence similarity to human CENP-B. 5' and 3' flanking and untranslated sequences were conserved at a level of 94.6% and 82.7%, respectively, suggesting that the regulatory properties of CENP-B may be conserved as well. CENP-B mRNA was detected in mouse cells and tissues and an immunoreactive nuclear protein identical in size to human CENP-B was detected in mouse 3T3 cells using human ACA. Analysis of the sequence of CENP-B revealed a segment of significant similarity to a DNA binding motif identified for the helix-loop-helix (HLH) family of DNA binding proteins. These data demonstrate that CENP-B is a highly conserved mammalian protein that may be a member of the HLH protein family and suggest that it plays a role in a conserved aspect of centromere structure or function.

PC12 cells stimulate slow-myosin light chain 2 synthesis in chick breast muscle culture.

Skeletal muscle fibers developing in vitro synthesize predominantly fast-myosin light chains, with a small contribution (less than 10%) from slow-myosin light chain 2. Muscle fibers can be cocultured with a rat adrenal pheochromocytoma-derived nerve cell line (PC12) known to display properties similar to sympathetic neurons. PC12 cells cultured alone synthesize catecholamines and respond to nerve growth factor by synthesizing acetylcholine and extending neurite structures. They also synthesize significant amounts of acetylcholine in the presence of nonneuronal cell types, including muscle. When cocultures of skeletal muscle fibers and PC12 cells are established, the muscle cells respond with an increased level of slow light chain 2 synthesis. Myosin light chains were identified by two-dimensional gel electrophoresis and immunoblotting with an antiserum specific to slow light chain 2.

Streptomycin retards the phenotypic maturation of chick myogenic cells.

As part of an effort to optimize conditions required for the complete maturation of muscle cells in vitro, we have investigated the effects of the antibiotics penicillin, streptomycin, and Fungizone (amphotericin B) on the development of cultured chick embryo skeletal muscle. It is shown that even low dosages of streptomycin, but not penicillin or Fungizone, retard protein synthesis and accumulation in these cultures. Myosin accumulation was also reduced and the appearance of striations in fused cells was delayed in myotubes formed in medium containing streptomycin. Additional data suggest that this overall retardation of myogenesis is due to the influence of streptomycin on maturing myotubes rather than early proliferation and cell fusion. These results are discussed with regard to recent efforts to promote the full maturation of muscle cells grown in culture.

Drainage of intraabdominal abscesses. A comparison of surgical and computerized tomography guided catheter drainage.

Over an 18 month period, 130 patients were sent to the radiology department for diagnostic CT scans to localize intraabdominal abscesses. Of these patients, 71 (55 percent) were diagnosed as having intraabdominal abscesses. Fifty-one surgical procedures and 17 radiologic drainage procedures were done. The cure rate of patients undergoing an operative procedure was 88 percent, and the major complication rate was 23 percent. The cure rate of patients undergoing CT guided catheter drainage was 47 percent and the complication rate was 6 percent. CT guided catheter drainage may be the initial treatment of choice in institutions that specialize in invasive procedures performed by radiologists. However, in institutions that have not concentrated their efforts on this problem, the results may not be as good, and it may not be wise to assume these data have general applicability. Greater cooperative efforts between departments of radiology and surgery should lead to better results in treatment of intraabdominal abscesses and better understanding of the best methods of treating them.

Human fetal muscle and cultured myotubes derived from it contain a fetal-specific myosin light chain.

Human fetal muscles at ages 110, 125, and 132 days contain a fetal-specific myosin light chain. This light chain is absent in adult human muscle, copurifies with myosin, and is identified as a slow light chain because it reacts with purified antibody to chicken slow muscle light chains and does not react strongly with antibody to fast myosin light chains. This light chain is synthesized in cultures of fetal muscle along with normal myosin light chains. The presence of a fetal light chain in culture provides a marker for studies of human muscle disease in which it is important to know when or if the muscle makes a transition from embryonic or fetal expression to true adult phenotype.