Somatostatin receptors 2 and 5 are preferentially expressed in proliferating endothelium.

Angiogenesis is characterised by activation, migration and proliferation of endothelial cells and is central to the pathology of cancer, cardiovascular disease and chronic inflammation. Somatostatin is an inhibitory polypeptide that acts through five receptors (sst 1, 2, 3, 4, 5). Sst has previously been reported in endothelium, but their role remains obscure. Here, we report the expression of sst in human umbilical vein endothelial cells (HUVECs) in vitro, during proliferation and quiescence. A protocol for culturing proliferating and quiescent HUVECs was established, and verified by analysing cell cycle distribution in propidium-iodide-stained samples using flow cytometry. Sst mRNA was then quantified in nine proliferating and quiescent HUVEC lines using quantitative reverse transcriptase-polymerase chain reaction. Sst 2 and 5 were preferentially expressed in proliferating HUVECs. All samples were negative for sst 4. Sst 1 and 3 expression and cell cycle progression were unrelated. Immunostaining for sst 2 and 5 showed positivity in proliferating but not quiescent cells, confirming sst 2 and 5 protein expression. Inhibition of proliferating cells with somatostatin analogues Octreotide and SOM230, which have sst 5 activity, was found (Octreotide 10(-10)-10(-6) M: 48.5-70.2% inhibition; SOM230 10(-9)-10(-6) M: 44.9-65.4% inhibition) in a dose-dependent manner, suggesting that sst 5 may have functional activity in proliferation. Dynamic changes in sst 2 and 5 expression during the cell cycle and the inhibition of proliferation with specific analogues suggest that these receptors may have a role in angiogenesis.

Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat.

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus.

Knee manipulation after total knee arthroplasty.

To determine if any factors are associated with knee stiffness after total knee arthroplasty (TKA), we retrospectively reviewed the medical records and radiographs of patients who had knee manipulation after total knee replacement at Scott & White Memorial Hospital from 1983 to 1993. Twenty-five patients who had knee manipulation after TKA were matched by surgeon, year of surgery, and age (+/- 5 years) with a study group of 25 patients who did not have knee manipulation after TKA. Patients in the manipulated group had decreased flexion at the time of discharge from the hospital after the knee arthroplasty and a decreased final flexion. The age of the patient, time from surgery to manipulation, and preoperative flexion did not correlate with final flexion attained in the manipulated group. Relative to the control study group, the manipulated group had an increase in postoperative anteroposterior femoral thickness. A decrease in patellar height was noted both in the manipulated group and in the control nonmanipulated group. There was no significant difference between groups for a change in patellar height.

Stanniocalcin 2: characterization of the protein and its localization to human pancreatic alpha cells.

Stanniocalcin (STC) is a hormone that was originally identified in fish, where it inhibits calcium uptake by the gills and gut and stimulates phosphate adsorption by the kidney. Recently, two mammalian homologues of stanniocalcin were identified. The first (STC1) shows 61% identity to the fish stanniocalcins and appears to have a function similar to that of the fish stanniocalcins. The second homologue (STC2) is 30-38% identical to the fish stanniocalcins, and is characterized by unique cysteine and histidine motifs that are not found in the other stanniocalcins. We purified both the native hamster and recombinant human STC2 proteins and obtained a partial amino acid sequence of the hamster protein. Both proteins behave as a disulfide bonded homodimer, which undergoes post-translational modification(s). The STC2 gene was localized to human chromosome 5q35. Northern blot analysis revealed that the primary site of human STC2 production is the pancreas, and immunostaining localized the STC2 protein to a subpopulation of cells in the islet. Double immunostaining for STC2 and either insulin or glucagon revealed that STC2 protein is found in the alpha cells, but not the beta cells. We speculate that STC2 may play a role in glucose homeostasis.

Photographic standards in plastic surgery.

Standard photographic technique in plastic surgery is an important topic that has been stressed in the discipline over the past several years. Clinical photographs should always be taken with the same camera lens, lens setting, lighting, film, and patient position to ensure reproducibility and to enable valid pre- and postoperative comparisons. A 35-mm single lens reflex camera is highly recommended for this type of photography. Two lenses are suggested, one with a focal length range of 50 to 60 mm and one with a focal length range of 90 to 105 mm. Both should have macro capability. Two or more flash units are recommended, either camera-mounted or a studio system set-up in the office. Using the patient preparation method and technique outlined in the text, the Standards in Clinical Photography achieve consistency from patient to patient and also in the same patient in pre- and postoperative photographs. Henceforth, the information discussed in the article forms the basis for standard views, regardless of the image-capture medium.

Isolation, characterization and baculovirus-mediated expression of the cDNA encoding cytosine DNA methyltransferase from Pisum sativum.

A series of overlapping clones complementary to the Arabidopsis cytosine-5 DNA methyltransferase (C-5 MTase) has been isolated from pea cDNA libraries. The assembled nucleic acid sequence contains an open reading frame of 4761 bp encoding a protein of 1554 amino acids. Like other eukaryotic C-5 MTases, the inferred protein has a presumed regulatory N-terminal region linked to a catalytic C-terminal domain, which has eight of the ten conserved motifs found in prokaryotic C-5 MTases. The pea C-5 MTase has 65% identity at the nucleotide level and 61% identity at the protein level, with the Arabidopsis C-5 MTase. The catalytic domain of the pea enzyme shares 78% identity with Arabidopsis and approximately 52% identity with murine and human C-5 MTases, including the relative position of the proline-cysteine dipeptides of the catalytic centre. Using the conserved region of the cDNA as a probe, we have identified a transcript of 5 kb. Southern blot analysis of pea genomic DNA with the above probe indicates the presence of a single gene. Using poly(A)+ RNA from different developmental stages and different tissues, we have observed that expression is confined mostly to the rapidly dividing tissues of the plant. Expression of this assembled cDNA in a baculovirus system gives a protein of approximately 174 kDa. The expressed protein can be cross-linked, in an AdoMet-dependent manner, to duplex oligonucleotide substrates containing FdC in place of target cytosines in either CG or CAG/CTG sequences.

Laser-photosensitizer assisted immunotherapy: a novel modality for cancer treatment.

Photosensitizer-enhanced laser treatment, where dyes are activated in situ by lasers of appropriate wavelengths, provides highly selective tissue destruction, both spatially and temporally, through photophysical reactions. Although laser-sensitizer treatment for cancer can achieve a controlled local tumor cell destruction on a large scale, total tumor eradication may not be accomplished because of the incomplete local tumor killing or the presence of tumor metastases, or both. The long-term control of cancer depends on the host immune surveillance and defense systems in which both cell-mediated and humoral responses are critical. In this study we report a novel minimally invasive cancer treatment combining the laser photophysical effects with the photobiological effects. Irradiation of a rat mammary tumor by an 805 nm diode laser, after an intratumor administration of a specific photosensitizer, indocyanine green in a glycated chitosan gel, caused immediate photothermal destruction of neoplastic cells. Concomitantly this treatment stimulated the immunological defense system against residual and metastatic tumor cells. Increases in survival rate and in the eradication of tumor burden, both primary and metastatic, were observed after this treatment. Furthermore, the resistance of successfully treated rats to tumor rechallenge demonstrated a long-lasting systemic effect of the treatment. These findings indicate that our treatment has triggered a specific humoral immune response in the tumor-bearing rats.

Spreading of methylation along DNA.

Mouse DNA methyltransferase is able to catalyse the transfer of a methyl group to certain CG-containing single-stranded oligonucleotides. The presence of a methylcytosine is required for efficient transfer. This methylcytosine may or may not be on the same oligonucleotide as that containing the accepting CG dinucleotide. When the accepting CG dinucleotide forms part of an unmethylated CG dinucleotide pair, its accepting activity is dramatically reduced. This provides the potential for methylation to spread along the DNA when it is rendered single-stranded at replication. It could also help to maintain fully methylated CG islands and asymmetrically methylated sites.

Photothermal effects on murine mammary tumors using indocyanine green and an 808-nm diode laser: an in vivo efficacy study.

Murine mammary tumors were treated using indocyanine green and an 808 nm diode laser, and the in vivo chromophore-enhanced photothermal effects on the tumor burden and on tumor rat survival were investigated. The power of the laser was selected in the range of 5-10 W, and irradiation duration 3-5 min. One percent aqueous indocyanine green solution in a volume of 100-200 microliters was administered in situ, either acutely or 24 h prior to the treatment. The photothermal interaction was apparent under all our treatment conditions with a well-defined spatial containment in this study and the tumor growth was slowed after treatment. The post-treatment observation showed tumor recurrence and metastasis; no long-term survival was achieved with the single application of laser in conjunction with indocyanine green. Our results pose a question on the efficacy of the photothermal interaction even though tumor cell destruction can be achieved in a large and controlled scale. However, this highly selective photothermal impact on the tumor tissue did suggest that this method be applied repeatedly to be more effective and be used as the precursor of other modalities, such as chemotherapy, radiation therapy, immunotherapy, and surgery.

DNA binding and methyl transfer catalysed by mouse DNA methyltransferase.

By using a purified fraction of mouse DNA methyltransferase we have shown, by gel-retardation analysis, that the enzyme forms a low-affinity complex preferentially with hemimethylated DNA; the complexes formed with unmethylated or with fully methylated DNA are of even lower affinity, and only very weak interaction occurs with DNA lacking CG dinucleotides. Interaction is inhibited by N-ethylmaleimide. Methyl transfer from S-adenosyl-methionine is associated with the release of the fully methylated product from the complex. Complexes formed with the intact enzyme are extremely large, but limited trypsin treatment allows a major complex to enter the gel. DNA binding is not inhibited by this limited proteolysis of the native enzyme.

Chromophore-enhanced in vivo tumor cell destruction using an 808-nm diode laser.

Rat mammary tumors were treated using an 808-nm diode laser in a power range of 3-15 W. Photothermolysis was selectively enhanced by the chromophore indocyanine green (ICG), which has an absorption peak corresponding to the laser wavelength. ICG, injected into neoplastic tissues 24 h before laser exposure, was retained in sufficient quantity to produce a strong photothermal reaction. With appropriate laser power and adequate irradiation duration, laser energy could inflict severe photothermal damage to the entire targeted tumor tissue while leaving the skin and other interdicted tissue undamaged. Higher laser powers (10-15 W) produced more surface damage that limited light transmission and as a result gave rise to reduced regions of thermal destruction. Post-treatment observation revealed the survival of numerous tumor cells. This finding questions the long term efficacy of the photothermal effect of a single treatment using the combination of the ICG and the diode laser, particularly in the absence of other modalities.

Chromophore-enhanced laser-tumor tissue photothermal interaction using an 808-nm diode laser.

A diode laser was used to irradiate tumor tissue, with indocyanine green as the chromophore. The 808-nm wavelength radiation falls within the absorption peak of the chromophore (about 780 nm). The preliminary results in this report revealed clear and significant coupling of this laser and indocyanine green in laser-tissue photothermal interaction. The chromophore targeted tissue showed laser damage while peripheral tissues remained intact. Without the chromophore, this laser inflicted no apparent tissue damage in the non-contact mode with irradiance up to 1755 J/cm2.

S-adenosylmethionine metabolism in herpes simplex virus type 2-infected cells.

Infection of primary rat embryo cells with herpes simplex virus type 2 has previously been reported to produce a dramatic and rapid inhibition of cellular DNA methylation. However, it has neither an immediate effect on S-adenosylmethionine breakdown nor on the relative pool sizes of S-adenosylmethionine and S-adenosylhomocysteine.

Vector methylation inhibits transcription from the SV40 early promoter.

Methylation of a plasmid containing the SV40 promoter linked to the chloramphenicol acetyl transferase (CAT) gene, with either murine DNA methylase or methylase SssI results in inhibition of the expression of the reporter gene after transfection into cultured cells. Methylation of the plasmid with the methylases HhaI and HpaII has no effect on the expression of this gene. Protein-DNA interactions in the SV40 promoter are not affected by the presence of methylcytosine suggesting that inactivation results from the formation of an inactive chromatin structure that is dependent on the high CG content of the plasmid.

Can flow cytometry reduce the workload for cervical screening? The results of a series of 622 specimens.

A simple method permitting the flow cytometric examination of cervical specimens has been developed and an assessment made of the feasibility of relying on this method to screen women for cervical neoplasia. Examination of four flow cytometric parameters showed differences between morphologically normal and abnormal specimens and allowed identification of a proportion of the normal specimens. The system had a false negative rate of 8%. Our experience with cervical specimens has revealed a number of problems associated with their examination by flow cytometry and these are discussed.

Kinetic aspects of Ki-67 antigen expression in a normal cell line.

Using a normal cell line derived from a human fetus, the disappearance and reappearance of the Ki-67-reactive antigen following modification of the cell cycle was observed and estimated immunohistologically. It was found that G1/G0 arrest induced by serum deprivation resulted in loss of the antigen in 24 h in all but a few (usually less than 10%) of cells. Return to normal medium and resumption of growth was accompanied by reappearance in 30 h. When entry into S-phase was prevented by desferrioxamine, reappearance of the antigen still occurred but only lasted for about 24 h. Inhibition of protein synthesis with cycloheximide also caused fading and eventual loss of immunostaining. In view of the ease with which this antigen becomes undetectable with cessation of protein synthesis and interruption of the cell cycle, we agree with those who advise caution in the use of Ki-67 to measure growth fraction in changeable cell populations such as tumours.

DNA demethylation in erythroleukaemia cells.

Despite a fall in the proportion of CGs methylated, evidence has not been obtained for significant demethylation of prelabelled DNA when mouse erythroleukaemia cells are induced to differentiate. There is, however, a delay in the methylation of the DNA that is synthesised in the early period of induction, leading to its undermethylation by 30-50% and this may be a contributory cause of the observed fall in CG methylation.

Mouse DNA methylase. Intracellular location and degradation.

DNA methylase extracted with low salt from mouse Krebs II ascites cell nuclei has been degraded stepwise by trypsin treatment. Degradation, accompanied by a limited reduction in size of the native enzyme, leads to the progressive introduction of several nicks so that, eventually, fragments of 14, 18, 24 and 28 kD are released on denaturation. This illustrates the domain structure of the enzyme. In contrast to ascites cell nuclear extracts, preparations from liver nuclei are already nicked and the major from of the enzyme contains a 100 kD fragment though the native molecular weight is unchanged. Newborn mouse liver contains more undegraded enzyme that is mostly firmly-bound within the nucleus. Trypsin treatment increases the de novo activity of the enzyme and prevents its aggregation in the absence of salt, even in the presence of high concentrations of native DNA.