Timing of cyclin D1 expression within G1 phase is controlled by Rho.

The expression of cyclin D1 in mid-G1 phase is associated with sustained ERK activity, and we show here that Rho is required for the sustained ERK signal. However, we also report that Rho inhibits an alternative Rac/Cdc42-dependent pathway, which results in a strikingly early G1-phase expression of cyclin D1. Thus, cyclin D1 is induced in mid-G1 phase because a Rho switch couples its expression to sustained ERK activity rather than Rac and Cdc42. Our results show that Rho is crucial for maintaining the correct timing of cyclin D1 expression in G1 phase and describe a new role for cytoskeletal integrity in the regulation of cell cycle progression.

Involvement of CD44 and its variant isoforms in membrane-cytoskeleton interaction, cell adhesion and tumor metastasis.

CD44s (standard form of CD44) is a transmembrane glycoprotein whose external domain displays extracellular matrix adhesion properties by binding both hyaluronic acid (HA) and collagen. The cytoplasmic domain of CD44s interacts with the cytoskeleton by binding directly to ankyrin. It has been shown that post-translational modifications, such as phosphorylation (by protein kinase C), acylation (by acyl-transferase) and GTP-binding enhanced CD44's interaction with cytoskeletal proteins. Most importantly, the interaction between CD44s and the cytoskeletal protein, ankyrin, is required for the modulation of CD44s cell surface expression and its adhesion function. Recently, a number of tumor cells and tissues have been shown to express CD44 variant (CD44v) isoforms. Using RT-PCR and DNA sequence analyses, we have found that unique CD44 splice variant isoforms are expressed in both prostate and breast cancer cell lines and carcinomas. Most importantly intracellular ankyrin is preferentially accumulated underneath the patched/capped structures of CD44 variant isoform in both breast and prostate cancer cells attached to HA-coated plates. We propose that selective expression of CD44v isoforms unique for certain metastatic carcinomas and their interaction with the cytoskeleton may play a pivotal role in regulating tumor cell behavior during tumor development and metastasis.

Interaction of CD44 variant isoforms with hyaluronic acid and the cytoskeleton in human prostate cancer cells.

CD44 is a glycosylated adhesion molecule which may undergo alternative splicing of 10 possible exons to generate variant isoforms. A number of CD44 variant isoforms expressed by tumor cells have been correlated with metastatic and proliferative behavior. In this study, we have characterized CD44 isoform expression on three prostate cancer cell lines: ALVA-31, PPC-1, and LNCaP. Using reverse transcriptase-polymerase chain reaction, we have found that ALVA-31 and PPC-1 cells express multiple CD44 isoforms, including CD44s (standard form), CD44E (epithelial form), and an exon 14-containing form. In addition, two smaller forms have been detected: one using an alternative donor splice site within exon 5, and a novel form omitting exon 5 entirely. The CD44 isoforms expressed by ALVA-31 and PPC-1 cells appear to be preferentially located on the cell surface. By contrast, LNCaP cells do not express any of the CD44 forms at the RNA or protein level. Both PPC-1 and ALVA-31 cells display tumorigenesis and invasiveness in nude mice, whereas LNCap cells exhibit a less malignant phenotype, suggesting a correlation between CD44 variant (CD44v) expression and aggressive prostate tumor behavior. Functional characterization reveals that CD44 mediates prostate cell adhesion to extracellular hyaluronic acid (HA). In addition, the CD44 cytoplasmic domain binds specifically to ankyrin, a membrane cytoskeletal protein. Double immunofluorescence labeling and confocal microscopic analyses indicate that HA binding induces the HA receptor (i.e., CD44) to form capped structures. Importantly, intracellular ankyrin is preferentially accumulated underneath HA receptor-capped structures. These results suggest that cytoskeletal proteins such as ankyrin are closely associated with CD44-mediated signaling events induced by HA. Finally, HA-mediated transmembrane interactions between CD44 isoforms and cytoskeletal proteins (i.e. ankyrin) may play a pivotal role in regulating tumor cell behavior during human prostate cancer development.

ADP-ribosylation factors: a family of approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin.

ADP-ribosylation factors (ARFs) comprise a family of approximately 20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of Gs alpha, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.

Isolation of recombinant ADP-ribosylation factor 6, an approximately 20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins, which, like other members of the ras superfamily, are activated by exchanging bound GDP for GTP and inactivated through hydrolysis of the gamma-phosphate of bound GTP to form GDP in a highly regulated cycle. ARF 6, a class III ARF, was expressed in Escherichia coli with its amino terminus fused to maltose-binding protein. Following release from maltose-binding protein, recombinant ARF 6 (rARF 6) exhibited maximal activity with or without GTP. Such constitutive activation was due to the predominance of ARF-GTP over ARF-GDP, as demonstrated by nucleotide analysis. rARF 6 expressed in E. coli without amino-terminal extension was bound primarily to GDP and exhibited typical GTP-dependent activity. After release from maltose-binding protein, rARF 6-GTP was stable; only a fraction of the nucleotide was removed using EDTA, whereas urea denaturation restored complete GTP dependence. [alpha-32P]GTP bound to rARF 6 was in part protected from hydrolysis by alkaline phosphatase and resulted in the formation of [alpha-32P]GTP, -GDP, and -GMP, whereas unbound nucleotide was completely hydrolyzed to guanosine. Thus, amino-terminal extension of rARF 6, by maltose-binding protein, promoted the formation of a constitutively activated GTP-bound species. By analysis of this species, we confirmed that rARF 6 lacks the intrinsic ability to hydrolyze bound GTP and speculate that maltose-binding protein may inhibit hydrolysis by extrinsic factors.

Application of maxillofacial miniplating and microplating systems to the hand.

During the past several years, a profound change in our approach to bone stabilization in the hand has occurred. Today, standard management of almost any fracture requiring open reduction as well as most arthrodeses and osteotomies will incorporate rigid fixation employing one of the maxillofacial systems. Many of the early concerns, such as increased complexity of the procedure, the need for extensive periosteal stripping, bulkiness of the plates (with potential palpation, tenderness, and the impingement on tendon and joint motion), and the anticipated frequent need for removal, have been allayed. Thinner plates, the smallest screws from the miniplating systems, and the introduction of the microplating systems (Luhr and Synthes) have obviated much of the anticipated difficulty with tendon gliding and have made the necessity for subsequent plate removal a rarity in our practice. The use of these systems allows much earlier mobilization postoperatively, reducing the incidence of joint stiffness and tendon adhesion. This paper discusses our series of 143 patients (average follow-up time over 18 months) and the application of miniplating and microplating systems for a variety of fractures, osteotomies, and arthrodeses in the hand.

Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells.

ADP-ribosylation factors (ARFs) are a family of highly conserved, 20-kDa guanine nucleotide-binding proteins that participate in protein trafficking and enhance cholera toxin-catalyzed ADP-ribosylation. ARF 2 from bovine retinal cDNA was expressed in Sf9 insect cells using recombinant baculovirus and compared to the major insect cell ARF (Sf9 ARF) and to recombinant ARF 2 expressed in Escherichia coli (E. coli rARF 2). The 150000g supernatant and particulate fractions of freeze-thawed, recombinant ARF 2 baculovirus-infected cells contained immunoreactive proteins of 20 and 21 kDa at significantly higher levels than were found in uninfected cells. Infected Sf9 cells incorporated [3H]myristate only into the 20-kDa protein. Sf9 cell recombinant ARF 2 (Sf9 rARF 2) and Sf9 ARF were separated by isoelectric focusing or ion-exchange chromatography and identified by microsequencing of HPLC-purified tryptic peptides. Sf9 ARF displayed considerable sequence identity to mammalian class I ARFs. Both Sf9 ARF and Sf9 rARF 2 stimulated in a GTP-dependent manner cholera toxin-catalyzed ADP-ribosylation. The Ka for GTP of Sf9 ARF was, however, significantly lower than that of Sf9 rARF 2 or E. coli rARF 2. Myristoylation did not significantly affect the ability of ARF 2 to enhance cholera toxin-catalyzed ADP-ribosylation or the Ka for GTP. Despite the sequence identities and the fact that both were synthesized in insect cells, the endogenous Sf9 ARF was functionally different from Sf9 rARF 2.

Inhibition of perioperative platelet aggregation using toradol (ketorolac).

Traditionally, aspirin is used as an inexpensive and usually well-tolerated agent to accomplish inhibition of platelet aggregation after microvascular surgery. Occasionally, however, aspirin is contraindicated. We have successfully used Toradol (ketorolac) after microvascular tissue transfer to inhibit platelet aggregation postoperatively, and documented this in our laboratory. We present two individuals on our surgical service requiring microvascular free tissue transfer with documented allergic reactions to aspirin. Platelet function was successfully suppressed while on the Toradol regimen, and this function returned to normal within 24 hours after stopping this therapy. We believe this agent may have some use for the microvascular surgeon for the population of patients in which aspirin is contraindicated or difficult to administer.

Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs). Molecular basis for differences in requirements for activity of mammalian ARFs.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase. Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid. Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified. To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized. Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity. Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate. In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP. rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding. These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.

Patterns of free-radical production after tourniquet ischemia: implications for the hand surgeon.

Since use of the pneumatic tourniquet is standard procedure for the hand surgeon, ischemic and reperfusion injury is a risk. To determine optimal periods of ischemia, 100 rabbit hindlimbs were subjected to various ischemic insults and analyzed for malondialdehyde (an indicator of free-radical production). Group 1 (3 hours of continuous ischemia) had 12.5 percent more reperfusion damage than controls (p less than 0.05). Group 2 (three 1-hour ischemic insults) had 10 percent more damage than controls (p less than 0.05). Group 3 (two 90-minute ischemic episodes) had 21 percent more damage than controls (p = 0.0001). Group 4 (4 1/2 hours of continuous ischemia) had 14.5 percent more damage than controls (p less than 0.01). Group 5 (three 90-minute ischemic episodes) had 10.8 percent more damage than controls (p less than 0.01). Group 6 (6 hours of continuous ischemia) had 17.5 percent more damage than controls (p less than 0.002). Group 7 (four 90-minute ischemic episodes) had 14 percent more damage than controls (p less than 0.01). Group 8 (three 2-hour ischemic episodes) had 22.5 percent more damage than controls (p less than 0.003). And group 9 (two 3-hour ischemic episodes) had 42 percent more damage than controls (p less than 0.0001). These results suggest a direct correlation in reperfusion injury with duration of tourniquet ischemia. Additionally, allowing specific reperfusion periods in some groups ultimately increased the amount of reperfusion injury.

Nipple reconstruction using the "inchworm" flap.

A new method of nipple reconstruction called the "inchworm" flap is described. The name comes from the manipulation method used to position the flap for nipple projection. Experience with over 50 patients and including 58 breasts has been reviewed. There was adequate followup for 36 breasts in which overall maintenance of projection was 78% for this technique. When used after submuscular expansion for breast reconstruction, maintenance of projection reached 89%. The technique of flap manipulation is described in detail. This flap appears to be a reliable method for obtaining moderate nipple projection, particularly in the submuscular expansion breast reconstruction cases. It is not recommended for use in TRAM flap cases.

Impedance plethysmography: a new method for continuous muscle perfusion monitoring.

Vigilant postoperative monitoring of the buried muscle flap is critical after free transfer because early diagnosis of vascular insufficiency is essential to allow prompt correction. We have identified a monitoring method utilizing needle electrodes and impedance plethysmography that gives a beat-to-beat representation of muscular perfusion. In 25 New Zealand White rabbits the gastrocnemius muscle was isolated on its vascular pedicle, and two intramuscular needle electrodes were placed. The instantaneous impedance changes of the muscle (corresponding to the pulsatile volume changes of perfusion) were measured and recorded. Using this representation of perfusion, an independent judge was able to correctly diagnose muscular ischemia 100 percent of the time (n = 25). Further, the judge was able to correctly distinguish the ischemia as arterial (n = 10) or venous (n = 10) in origin 100 percent of the time. Additionally, we monitored muscle perfusion transcutaneously in five free muscle flaps and demonstrated a reliable impedance signal that correlated with perfusion.

Noninvasive assessment of microvessels with the duplex scanner.

This study was undertaken to determine if a duplex scanner equipped with a new 10 MHz probe could accurately evaluate microvascular anastomotic patency. The overall predictive accuracy of the duplex scanner was 90% (p less than 0.0001) with no difference noted among the three main anastomotic groups examined--acute artery, acute vein, and long-term artery. There was, however, a statistically significant difference (p less than 0.05) in the ability to interpret vessels that were patent (100%) versus those that were partially occluded (73%) or occluded (88%). It is believed that the duplex scanner has potential applications to preoperatively and intraoperatively study microvessels and postoperatively to supplement other techniques in monitoring acute and long-term anastomotic patency.