Polyubiquitylation of cellular proteins has long been recognized as a prelude to a degradative fate in proteasomes. In recent years, however, ubiquitin conjugation has emerged as a regulatory strategy of considerable versatility. Most notably, monoubiquitylation is attributed an intimate role in trafficking of membrane proteins between various cellular compartments. Diverse classes of transmembrane proteins from across the eukaryotic spectrum (e.g., epidermal growth factor-receptor and other receptor tyrosine kinases) become modified with monoubiquitin molecules. Monoubiquitylation of substrates, in turn, regulates both their endocytosis at the plasma membrane and sorting in endosomes for delivery to lysosomes or vacuoles. A mechanistic rationale lies in the identification of a growing list of ubiquitin-binding domains carried by a variety of endocytic adaptor proteins. Thus, ubiquitin-conjugated membrane proteins may form extensive contacts with the endocytic machinery. Further, ubiquitin-binding adaptors and other endocytic components are, likewise, often monoubiquitylated. In this case, ubiquitin conjugation may serve to enhance intermolecular avidity in cargo-bound endocytic complexes, or alternatively, to mediate timely inactivation of ubiquitin-binding adaptors. Interestingly, the ubiquitin/endocytosis interface is appropriated by pathogenic organisms, for instance, during budding of viruses from host-infected cells. Moreover, compromised ubiquitin-mediated transport of certain signaling receptors is associated with disease states, including oncogenic transformation.

We describe a unique E3, the F-box protein, Ufo1, of yeast. Ufo1 recruits the mating switch endonuclease, Ho, to the SCF complex for ubiquitylation. In addition to the F-box and WD40 protein-protein interaction domains found in all F-box proteins, Ufo1 has a unique domain comprising multiple copies of the ubiquitin-interacting motif. Ufo1 interacts with the UbL-UbA protein, Ddi1, via its UIMs[1], and this is required for turnover of SCF ^Ufo1 complexes. This is a novel function for an UbL-UbA protein. Deletion of the genomic UFO1 UIMs is lethal and our data indicate that Ufo1ΔUIM acts as a dominant negative leading to inhibition of the SCF pathway of substrate degradation and to cell cycle arrest. Furthermore, we found that Ddi1 is required for the final stages of degradation of Ho endonuclease. In the absence of Ddi1, Ho does not form a complex with the 19S RP and is stabilized. Stabilization of Ho leads to perturbation of the cell cycle and to the formation of multi-budded cells. Our experiments uncover a novel role for the ubiquitin-proteasome system in maintenance of genome stability.

The Ink4a-Arf locus, which encodes two distinct tumor suppressor proteins, is inactivated in many cancers. Whereas p16^Ink4a is an inhibitor of cyclin D-dependent kinases, p19^Arf (p14^ARF in humans) antagonizes the E3 ubiquitin protein ligase activity of Mdm2 to activate p53. We now recognize that Arf functions in both p53-dependent and -independent modes to counteract hyper-proliferative signals originating from proto-oncogene activation, but its p53-independent activities remain poorly understood. Arf proteins are highly basic (> 20% arginine content, pI > 12) and predominantly localize within nucleoli in physical association with an abundant acidic protein, nucleophosmin (NPM/B23). When bound to NPM[1], Arf proteins are relatively stable with half-lives of 6–8 hours. Although mouse p19^Arf contains only a single lysine residue and human p14^ARF has none, both proteins are N-terminally ubiquitinated and degraded in proteasomes. Through as yet uncharacterized mechanisms, p19^Arf induces p53-independent sumoylation of a variety of cellular target proteins with which it interacts, including both Mdm2 and NPM. A naturally occurring NPM mutant (NPMc) expressed in myeloid leukemia cells redirects both wild-type NPM and p19^Arf to the cytoplasm, inhibits Arf-induced sumoylation, and attenuates p53 activity. Thus, ubiquitination and sumoylation can each influence Arf tumor suppressor activity.

Background: Virtual reality immersion has been advocated as a new effective adjunct to drugs for pain control. The attenuation of pain perception and unpleasantness has been attributed to the patient's attention being diverted from the real, external environment through immersion in a virtual environment transmitted by an interactive 3-D software computer program via a VR[1] helmet.

Objectives: To investigate whether VR immersion can extend the amount of time subjects can tolerate ischemic tourniquet pain.

Methods: The study group comprised 20 healthy adult volunteers. The pain was induced by an inflated blood pressure cuff during two separate, counterbalanced, randomized experimental conditions for each subject: one with VR and the control without VR exposure. The VR equipment consisted of a standard computer, a lightweight helmet and an interactive software game.

Results: Tolerance time to ischemia was significantly longer for VR conditions than for those without (P < 0.001). Visual Analogue Scale (0–10) ratings were recorded for pain intensity, pain unpleasantness, and the time thought about pain. Affective distress ratings of unpleasantness and of time thought about pain were significantly lower during VR as compared with the control condition (P < 0.003 and 0.001 respectively).

Conclusions: The VR method in pain control was shown to be beneficial. The relatively inexpensive equipment will facilitate the use of VR immersion in clinical situations. Future research is necessary to establish the optimal selection of clinical patients appropriate for VR pain therapy and the type of software required according to age, gender, personality, and cultural factors.

Background: Physicians in the community work on a tight and often pressured schedule; verbal and non-verbal techniques to terminate the patient-physician encounter are therefore necessary.

Objectives: To characterize ways of terminating the encounter.

Methods: Using a structured questionnaire we observed seven family physicians and nine consultants and recorded patient-physician encounters to assess techniques for terminating the encounter.

Results: In all, 320 encounters were recorded, 179 (55.9%) by consultants and 141 (44.1%) by family physicians. The mean duration of the encounters was 9.02 ± 5.34 minutes. The mean duration of encounters with family physicians was longer than consultants (10.39 vs. 7.93 minutes, P < 0.001). In most cases the encounter ended with the patient receiving printed documentation from the physician (no difference between family physicians and consultants). Consultants were more likely to end the encounter with a positive concluding remark such as “feel good” or “be well” (P < 0.01). There was no single occasion where termination of the encounter was initiated by the patient.

Conclusions: Giving a printed document to the patient appears to be perceived by both patients and physicians as an accepted way to end an encounter. Another good way to end the encounter is a positive greeting such as “feel good” or “be well.”
 

Background: The evaluation of influenza vaccine activity and potency are based on the immune response to hemagglutinin, and protection is indicated when a ≥ 1:40 titer of hemagglutination inhibition serum antibody is present. Neuraminidase, the second surface glycoprotein, may also have a role in protection, but little information on the immunologic response to this component is available.

Objectives: To determine whether any response to neuraminidase is evoked by intranasal immunization with a novel, whole, inactivated anti-influenza vaccine.

Methods: This study was part of a more comprehensive study of mucosal and serum immune response to this vaccine. Fifty-four young adults were immunized intranasally, 9 intramuscularly and 18 received a placebo. Twenty-three elderly people were immunized intramuscularly, and 21 elderly and 17 children were immunized intranasally. Serum and nasal antibodies to antigens N1 and N2 were determined by the lectin neuraminidase test.

Results: Serum response following intranasal vaccination was lower than after intramuscular vaccination, and ranged from 21.4 to 35.3% and 33.3 to 64.7% following intranasal vaccination and 52.2 to 77.8% and 47.8 to 88.9% after intramuscular vaccination, to N1 and N2 respectively. Nasal antibody response was low and was found only after intranasal vaccination, and response to N2 was better than to the N1 antigen.

Conclusions: It may be beneficial if future vaccines would include competent hemagglutinin and neuraminidase, which would afford a higher level of protection.
 

Background: The contribution of the abnormal DNA mismatch repair system to non-small cell lung cancer tumorigenesis is controversial and has not been reported in Jewish Israeli patients. Similarly, the involvement of 3p deletions in NSCLC[1] in the same population has not been assessed.

Objectives: To assess the contribution of the DNA-MMR[2] system to NSCLC pathogenesis by analyzing microsatellite instability, and evaluate loss of heterozygosity at 3p rates in Israeli NSCLC patients.

Methods: Paired DNA from tumorous and non-tumorous tissue was extracted, and genotyping for MSI[3] determination was carried out using the five Bethesda markers and for determining LOH[4] two 3p markers were used. Genotyping was performed using polymerase chain reaction amplification and size separation on an ABI semiautomatic DNA sequencer, and the allelic patterns of tumorous and non-tumorous tissue were compared.

Results: Forty-four NSCLCs from 35 smokers and 9 non-smokers were analyzed, with 26 of the 44 (59%) at stage I disease. Using five microsatellite markers (D17S250, D5S346, D2S123, BAT-25, BAT-26) (known as Bethesda markers) for MSI determination, 6 of the 44 tumors (13.6%) exhibited MSI in at least one marker. Similarly, genotyping for LOH at chromosome 3p was performed using two markers (D3S4103, D3S1234) located at 3p14.2 l. With D3S4103, 33 of the 44 patients successfully analyzed were homozygous and therefore non-informative with respect to LOH. Using D3S1234, 33 of 36 patients (91.7%) were heterozygous, and 23 of these individuals' tumors (69.7%) displayed LOH. Unexpectedly, 4 of 33 tumors (12.1%) genotyped by D3S4103, and 16 of 36 tumors (44.5%) genotyped by D3S1234 showed a pattern of MSI, even though only one of these tumors showed a similar pattern when genotyped with the five consensus markers. Overall, 23 of 44 tumors (52.3%) demonstrated MSI on at least one marker, and 5 of these 23 tumors (21.7%) had MSI on two or more markers.

Conclusions: MSI using 3p markers and not the Bethesda markers occurs at a high rate and in early stages in Jewish NSCLC patients.

Background: Metastatic melanoma is an aggressive and highly malignant cancer. The 5 year survival rate of patients with metastatic disease is less than 5% with a median survival of only 6–10 months. Drugs like dacarbazin (DTIC) as a single agent or in combination with other chemotherapy agents have a response rate of 15–30%, but the duration of response is usually short with no impact on survival. Interleukin-2-based immunotherapy has shown more promising results. The National Institutes of Health recently reported that lymphodepleting chemotherapy, followed by an adoptive transfer of large numbers of anti-tumor specific tumor-infiltrating lymphocytes, resulted in an objective regression in 51% of patients.

Objectives: To introduce the TIL[1] technology to advanced metastatic melanoma patients in Israel.

Methods: We generated TIL cultures from tumor tissue, choosing those with specific activity against melanoma and expanding them to large numbers.

Results: TIL cultures from nine patients were established and examined for their specific activity against the patients' autologous tumor cells. Twelve TIL cultures derived from 5 different patients showed the desired anti-tumor activity, making those 5 patients potential candidates for the therapy.

Conclusions: Pre-clinical studies of the TIL technology in a clinical laboratory set-up were performed successfully and this modality is ready for treating metastatic melanoma patients at the Sheba Medical Center's Ella Institute.

Background: We recently published preliminary evidence on the effectiveness of hypertonic saline in infants with viral bronchiolitis.

Objective: To further establish the efficacy of nebulized hypertonic saline in these infants

Methods: In a continuing, second-year randomized, double-blind controlled trial, an additional 41 infants (age 2.6 ± 1 months) hospitalized with viral bronchiolitis were recruited during the winter of 2001–2002. The infants received inhalation of 1.5 mg epinephrine dissolved either in 4 ml normal (0.9%) saline (Group I, n=20) or 4 ml hypertonic (3%) saline (Group II, n=22). The therapy was repeated three times daily until discharge. Pooling our 2 years of experience (2000–2002), a total of 93 hospitalized infants with viral bronchiolitis were recruited; 45 were assigned to Group I and 48 to Group II.

Results: The clinical scores at baseline were 7.6 ± 0.7 for Group I vs. 7.4 ± 1.3 for Group II (P = NS). However, the clinical scores at days 1 and 2 after inhalation differed significantly between the two groups, invariably favoring Group II: 7 ± 1 vs. 6.25 ± 1.1 (P < 0.05), 6.45 ± 1 vs. 5.35 ± 1.35 (P < 0.05), respectively. Adding aerosolized 3% saline to 1.5 mg epinephrine reduced the hospitalization stay from 3.5 ± 1.7 days in Group I to 2.6 ± 1.4 in Group II (P < 0.05). The pooled data of both years revealed that adding 3% saline to the inhalation mixture decreased hospitalization stay from 3.6 ± 1.6 to 2.8 ± 1.3 days (P < 0.05).
Conclusions: This second-year experience and our 2 year pooled data analysis strengthen the evidence that the combination of 3% saline/1.5 mg epinephrine benefits hospitalized infants with viral bronchiolitis

Background: Peritoneal dialysis is a widely accepted route for renal replacement. With the advent of endoscopy, many surgical techniques for the prevention of catheter failure have been proposed.

Objectives: To evaluate the outcomes of patients undergoing laparoscopic Tenckhoff catheter implantation, using the pelvic fixation technique.

Methods: Data analysis was retrospective. All procedures were performed under general anesthesia. A double-cuffed catheter was inserted using two 5 mm trocars and one 10 mm trocar, fixing its internal tip to the dome of the bladder and its inner cuff to the fascia. Catheter failure was defined as persistent peritonitis/exit-site/tunnel infection, severe dialysate leak, migration or outflow obstruction.

Results: LTCI[1] was performed in 34 patients. Mean patient age was 65 ± 17 years. In 12 of the 34 patients the indication for LTCI was end-stage renal failure combined with NYHA class IV congestive heart failure. Operative time was 35 ± 15 minutes. A previous laparotomy was performed in 9 patients. Hospital stay was 1.5 ± 0.6 days. The first continuous ambulatory peritoneal dialysis was performed after 20 ± 12 days. Median follow-up time was 13 months. There were several complications, including 5 (14%) exit-site/tunnel infections, 27 episodes (0.05 per patient-month) of bacterial peritonitis, 3 (9%) incisional hernias, 1 case of fatal intraabdominal bleeding, 2 (5.8%) catheter migrations (functionally significant), and 10 (30%) cases of catheter plugging, 8 of which were treated successfully by instillation of urokinase and 2 surgically. A complication-mandated surgery was performed in 8 patients (23.5%). The 1 year failure-free rate of the catheter was 80.8%. One fatal intraabdominal bleeding was recorded.
Conclusions: LTCI is safe, obviating the need for laparotomy in high risk patients. Catheter fixation to the bladder may prevent common mechanical failures