Creation of an artificial bifunctional intein by grafting a homing endonuclease into a mini-intein

The majority of inteins are comprised of a protein splicing domain and a homing endonuclease domain. Experimental evidence has demonstrated that the splicing domain and the endonuclease domain in a bifunctional intein are largely independent of each other with respect to both structure and activity. Here, an artificial bifunctional intein has been created through the insertion of an existing homing endonuclease into a mini-intein that is naturally lacking this functionality. The gene for I-CreI, an intron-encoded homing endonuclease, was grafted into the monofunctional Mycobacterium xenopi GyrA intein at the putative site of the missing endonuclease. The resulting fusion protein was found to be capable of protein splicing similar to that of the parent intein. In addition, the protein demonstrated site-specific endonuclease activity that is characteristic of the I-CreI homing endonuclease. The function of each domain therefore remained unaffected by the presence of the other domain. This artificial fusion of the two domains is a potential novel mobile genetic element.     

Protein splicing of the Deinococcus radiodurans strain R1 Snf2 intein

Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues.     

Interleukin-7 receptor expression and activation in nonhaematopoietic neoplastic cell lines

The high-affinity human interleukin-7 (IL-7)R is a heterodimeric complex consisting of the IL-7Ralpha and common interleukin-2 receptor gamma (IL-2Rgamma(c)) chains. Activation of the IL-7R complex is associated with tyrosine and serine residue phosphorylation of a number of intracellular substrates leading to proliferation and induction of various cellular differentiation processes. In this study, we demonstrate, by S1 nuclease protection assay, immunoprecipitation and in vitro kinase assay that functional human (h) IL-7R is expressed in haematopoietic and nonhaematopoietic cell lines. The National Cancer Institute (NCI) tumour panel of 60 cell lines (NCI60) was screened for the expression of IL-7R mRNA by S1 nuclease protection assay, and IL-7R mRNA was detected in 9 of 12 leukemia, 3 of 7 lung, 4 of 6 CNS, 2 of 7 melanoma, 2 of 7 renal, 1 of 6 colon and 1 of 6 breast cancer cell lines. Immunoblot analysis of haematopoietic, lung cancer and brain tumour cell lines demonstrated expression of IL-7R, IL-2Rgamma(c) and p59 fyn, suggesting that the components of an IL-7R signalling network are present in nonhaematopoietic neoplastic cells. Immunoprecipitation of IL-7Ralpha followed by an in vitro kinase assay demonstrated functional receptor phosphorylation events in the lung cancer cells but not in the brain tumour cell lines. The expression of functional IL-7R on epithelial tumour cells may represent a potential target for receptor-directed therapy.     

Diet-induced obesity and hepatic gene expression alterations in C57BL/6J and ICAM-1-deficient mice

The effects of high-fat feeding on the development of obesity were evaluated in intercellular adhesion molecule-1 (ICAM-1) knockout and C57BL/6J (B6) male mice fed a high-fat diet for < or =50 days. Serum and tissues were collected at baseline and after 1, 11, and 50 days on the diet. After 11 days on the diet, ICAM-1-deficient, but not B6, mice developed fatty livers and showed a significant increase in inguinal fat pad weight. At day 50, ICAM-1-deficient mice weighed less, and their adiposity index and circulating leptin levels were significantly lower than those of B6 controls. To better understand the early differential response to the diet, liver gene expression was analyzed at three time points by use of Affymetrix GeneChips. In both strains, a similar pattern of gene expression was detected in response to the high-fat diet. However, sterol regulatory element-binding protein-1, apolipoprotein A4, and adipsin mRNAs were significantly induced in ICAM-1-deficient livers, suggesting that these genes and their associated pathways may be involved in the acute diet response observed in the knockout mice.     

Therapeutic efficacy of melanoma-reactive TIL injected in stage III melanoma patients

Adoptive therapy for cancer using tumor-infiltrating lymphocytes (TIL) has mainly been investigated in cancer patients with advanced stage disease. The limited clinical success has not been encouraging, although this might be explained by poor TIL specificity and/or high tumor burden. To re-evaluate the effectiveness of adoptive therapy, we analyzed the capacity of tumor-reactive TIL injection in preventing the further development of disease in stage III melanoma patients after complete tumor resection. A phase II/III randomized trial was performed on 88 melanoma patients, who received autologous TIL plus interleukin-2 (IL-2) or IL-2 only. The duration of relapse-free survival was analyzed, taking into account the immunological specificity of injected TIL and the number of metastatic lymph nodes removed before treatment. Kaplan-Meyer analysis revealed that the injection of tumor-reactive TIL was statistically correlated with prolonged relapse-free survival in patients with only one metastatic lymph node. Therefore, improved clinical outcome could be obtained after adoptive therapy by selecting appropriate groups of patients and monitoring the specificity of the injected TIL populations.     

Optical tweezers in biology and medicine

Optical trapping techniques provide unique means to manipulate biological particles such as virus, living cells and subcellular organelles. Another area of interest is the measurement of mechanical (elastic) properties of cell membranes, long strands of single DNA molecule, and filamentous proteins. One of the most attractive applications is the study of single motor molecules. With optical tweezers traps, one can measure the forces generated by single motor molecules such as kinesin and myosin, in the piconewton range and, for the first time, resolve their detailed stepping motion.     

New perspectives of locomotor rehabilitation after stroke

The task-oriented approach incorporating treadmill walking for retraining gait early after stroke has contributed to promote locomotor recovery. To augment practice, training strategies such as mental practice and training in virtual environments are proposed. While the former offers more practice with less physical exertion, the latter allows safe practice in a variety of challenging environments. Work is under way to assess whether these new strategies can further enhance locomotor recovery.     

The role of magnetic resonance imaging in the management of vascular malformations of the trunk and extremities

Vascular malformations can usually be diagnosed on clinical grounds. They have a well-defined appearance on magnetic resonance imaging, which can effectively determine their tissue and flow characteristics. However, the role of cross-sectional imaging in the management of vascular malformations is not well defined. Most reviews suggest that magnetic resonance imaging should be reserved for cases in which the extent of the lesion cannot be estimated on physical examination. However, to date no group has compared the accuracy of physical examination alone to that of magnetic resonance imaging in determining this extent. A review was performed of all the patients evaluated for vascular malformations at the New York University Trunk and Extremity Vascular Anomalies Conference between July of 1994 and August of 1999. Patients who underwent magnetic resonance evaluation at other institutions and whose images were not available for review were excluded. All study patients either underwent magnetic resonance imaging examination at New York University Medical Center or had outside films reviewed at the center. The physical examination findings were compared with the magnetic resonance findings and the surgeon and radiologist made a joint decision about whether there was a correlation between the magnetic resonance and physical examination findings. Fifty-eight patients met the study criteria, 44 (76 percent) of whom were found to have more extensive disease on magnetic resonance examination than appreciated on physical examination. Of the 51 patients with low-flow vascular malformations (venous vascular malformations, lymphatic malformations, and capillary malformations), 39 (76 percent) had more extensive disease on magnetic resonance examination than on physical examination. Of the seven patients with high-flow arteriovenous malformations, five had more extensive disease on magnetic resonance. In all of the 44 patients whose magnetic resonance imaging findings did not correlate with those of the physical examination, therapeutic decision making was affected. Contrary to the conventional wisdom of published reviews, physical examination findings significantly underestimated the extent of vascular malformations in the majority of cases. Magnetic resonance imaging should be performed in all patients with vascular malformations of the trunk and extremities before therapy is planned. In an age when physicians are asked to justify their decisions, especially where the use of expensive diagnostic modalities is concerned, the situations in which these tests are indispensable must be clearly defined or else patients will be denied access to them.     

Identification of Naegleria fowleri in domestic water sources by nested PCR

The free-living amoeboflagellate Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to N. fowleri and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for N. fowleri by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for N. fowleri by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for N. fowleri were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home.     

Prenatal diagnosis of vascular anomalies: update and review of the literature

Optimal care for a subgroup of infants with complicated vascular anomalies requires prenatal diagnosis. Fetal vascular lesions are either vascular tumors or vascular malformations, both of which are often detected on routine ultrasound. Imaging, such as ultrasound and fetal MRI, can be used to examine lesions and provide the data for a differential diagnosis, which may impact the course of care both in utero and postnatally. Prenatal diagnosis provides the opportunity for antenatal intervention, parental counseling, and planning of the mode and location of delivery to optimize postnatal care. Prenatal diagnosis of vascular lesions also serves to alert the physician to the potential for associated syndromes and complications. Any indication of a vascular anomaly should be referred for further examination by an experienced multidisciplinary team of physicians to ensure the window in which evaluation, planning, and treatment can take place is not missed.