Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts

The hammerhead ribozyme is a small catalytic RNA molecule. Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a putative cleavage site within the target molecule. We have investigated the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin is a 347 kDa glycoprotein that is a major constituent of the elastin-associated microfibrils. Mutations in the FBN1 gene are responsible for Marfan syndrome (MFS), a common systemic disorder of the connective tissue. Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion, raising the possibility that reduction of the amount of product from the mutant FBN1 allele might be a valid therapeutic approach for MFS. A trans-acting hammerhead ribozyme (FBN1-RZ1) targeted to the 5' end of the human FBN1 mRNA has been designed and synthesized, and shown to cleave its target efficiently in vitro. FBN1-RZ1 cleavage is magnesium dependent and efficient at both 37 and 50 degrees C. Delivery of the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, by receptor- mediated endocytosis of a ribozyme-transferrin-polylysine complex, specifically reduces both cellular FBN1 mRNA and the deposition of fibrillin in the extracellular matrix. These results suggest that the use of hammerhead ribozymes is a valid approach to the study of fibrillin gene expression and possibly to the development of a therapeutic approach to MFS.      

Visual field deficits in cats reared with cyclodeviations of the eyes

Summary Visual fields of ten cats which had one or both eyes rotated at 8 days of age were measured by two forms of perimetry and compared to visual fields of two normal cats and of four cats with monocular rotations at 16 days, 3 months or 6 months of age. All animals showed excellent localization of visual stimuli and responded to the actual location of stimuli in space rather than to the retinal locus normally associated with that location. In cats with monocular rotations, the field of the normal eye was always normal, extending from 90 ° ipsilateral to 30 ° contralateral. Cats with rotations of one eye at 3 or 6 months of age had essentially normal fields in the rotated eye as well, while cats with surgery at 8 or 16 days had restricted horizontal fields. They responded only to stimuli in the ipsilateral hemifield; they were blind in the contralateral hemifield. Their superior and inferior visual fields were normal. The field deficits related consistently to visual field coordinates and not to the angle or direction of rotation. In cats with binocular rotations the visual field of at least one eye extended across the midline. Thus, the extent of the field depended upon sensorimotor experiences of the cat both before and after surgery. It is argued that these monocular field deficits have a central origin, not a retinal one.When tested with both eyes open, seven of 14 experimental animals did not respond throughout the visual field seen by each eye alone. The total visual field with both eyes open was less than the sum of the two monocular fields; greatest losses were most pronounced in the extreme periphery of the field ipsilateral to the rotated eye. Since changes in eye position (e.g., convergence during bincocular viewing) were not observed, it is suggested that the binocular losses indicate suppression of the deviated eye which has a central origin.All animals were tested for visual following, visually-triggered extension (placing), and visually-guided reaching. Cats which had been routinely encouraged to use the rotated eye(s) by occlusion of the other eye showed skilful performance within a few weeks after surgery as previously reported by Peck and Crewther (1975), Mitchell et al. (1976) and others. In contrast, two cats reared with both eyes open after unilateral rotation in infancy were profoundly handicapped, as previously reported by Yinon (1975, 1976).This research was supported by Grant NS 14116 from the US Public Health Service     

A structural analysis of the carbohydrate side chains on class I and class II histocompatibility antigens of the swine facilitated by heteroantisera specific for the denatured polypeptides

Rabbit heteroantisera specific for the denatured glycoprotein subunits of swine class I and class II major histocompatibility complex (MHC) antigens have been prepared and utilized to monitor changes in the mobilities of these polypeptides on SDS-polyacrylamide gel electrophoresis subsequent to various deglycosylation procedures. This information, in combination with lectin reactivity patterns for the glycoproteins bound to nitrocellulose, has made it possible to define specific structural features of the MHC antigen-associated carbohydrate side chains. Both the class I heavy (alpha) chain and the class II light (beta) chain bear a single, N-linked, complex-type oligosaccharide which reacts with lentil lectin (LcH), but not concanavalin A (Con A); a reactivity pattern suggesting the possibility of a special triantennary structure. In contrast, the class II heavy (alpha) chains appear to possess two carbohydrate units, one an N-linked, LcH-reactive, complex-type side chain, and the other, an N-linked, Con A-reactive, high-mannose-type of oligosaccharide. The data suggest considerable homology between the swine and human MHC antigens with respect to the structure of their carbohydrate side chains. The analysis also serves to illustrate how antibodies specific for the denatured polypeptide backbone of individual glycoproteins, along with lectin reactivity patterns, can be used to extract structural information about the attached carbohydrate moieties using minimal amounts of partially purified glycoproteins.     

Adjuvant-independent immunization by immunotargeting antigens to MHC and non-MHC determinants in vivo.

Using avidin as a model protein antigen, and biotinylated monoclonal antibodies as a convenient means of forming stable complexes with avidin, we have investigated the adjuvant-independent immunization of three mouse strains, C57BL/6, C3H and (C57BL/6 x C3H)F1, with immunoconjugates targeted to different class II MHC and non-MHC sites. The results confirm the effectiveness of anti-I-Ak and anti-I-Ab immunoconjugates with respect to priming for secondary IgG responses in (H-2b x H-2k)F1 mice, while indicating a lack of response in strains which are homozygous for the targeted allele. In terms of non-MHC targets in the monocyte-macrophage lineage, neither anti-MAC-1 nor anti-MAC-2 immunoconjugates were effective in any of the three strains. However, the 33D1 anti-dendritic cell antibody gave significant responses in all three strains, with the F1 response being more than 10-fold greater than the anti-class II immunoconjugates in either strain. These findings indicate that immunotargeting a protein antigen to a non-MHC determinant on dendritic cells in vivo can be an effective means of inducing an adjuvant-independent serological response, and that this approach can have significant advantages over anti-class II MHC immunotargeting.     

Fallopian tube necrosis after postpartum sterilization

Because it usually presents with nonspecific symptoms and occurs rarely, the diagnosis of fallopian tube torsion and necrosis is usually done at laparotomy. A 32 year-old woman returned to the hospital with right lower quadrant pain three days after a postpartum tubal sterilization procedure. Clinical, laboratory and imaging findings did not assist with the diagnosis. At laparotomy, after dissection of adhesions, a necrotic right fallopian tube was found. A salpingectomy was performed and the patient had an uneventful postoperative course. Fallopian tube torsion should be included in the differential diagnosis of pelvic pain in women. This patient has a good prognosis.     

Protective cytotoxic T lymphocyte responses against paramyxoviruses induced by epitope-based DNA vaccines: involvement of IFN-gamma

Plasmid DNA vectors have been constructed with minigenes encoding a single cytotoxic T lymphocyte (CTL) epitope from either the M2 protein of respiratory syncytial virus (RSV) or from the nucleoprotein of measles virus (MV) with or without a signal sequence (also called secretory or leader sequence). Following intradermal immunization, plasmids in which the CTL epitopes were expressed in-frame with the signal sequence were more effective at inducing peptide- and virus- specific CTL responses than plasmids expressing CTL epitopes without the signal sequence. This immunization resulted in protection against MV-induced encephalitis and a significant reduction in viral load following RSV challenge. The reduction of viral load following RSV challenge was abrogated by prior injection with anti-IFN-gamma antibodies. These results highlight the ability of epitope-based DNA immunization to induce protective immune responses to well-defined epitopes and indicate the potential of this approach for the development of vaccines against infectious diseases.      

Folate pathway gene alterations in patients with neural tube defects

Periconceptional folate supplementation reduces the recurrence and occurrence risk of neural tube defects (NTD) by as much as 70%, yet the protective mechanism remains unknown. Inborn errors of folate and homocysteine metabolism may be involved in the aetiology of NTDs. Previous studies have demonstrated that both homozygosity for the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, and combined heterozygosity for the C677T and for another mutation in the same gene, the A1298C polymorphism, represent genetic risk factors for NTDs. In an attempt to identify additional folate related genes that contribute to NTD pathogenesis, we performed molecular genetic analysis of folate receptors (FRs). We identified 4 unrelated patients out of 50 with de novo insertions of pseudogene (PS)-specific mutations in exon 7 and 3'UTR of the FRalpha gene, arising by microconversion events. All of the substitutions affect the carboxy-terminal amino acid membrane tail, or the GPI anchor region of the nascent protein. Furthermore, among 150 control individuals, we also identified one infant with a gene conversion event within the FRalpha coding region. This study, though preliminary, provides the first genetic association between molecular variations of the FRalpha gene and NTDs and suggests that this gene can act as a risk factor for human NTD.     

Low iron intakes among young women in Britain

A large survey by the Ministry of Agriculture, Fisheries and Food of people aged 15 to 25 showed that the women, and especially the participants "on a diet" or "watching their weight," generally had iron intakes well below the recommended daily allowance. Reduced iron intake appeared to result from diets of reduced iron concentration as well as from energy restriction. Further research is needed to establish whether this population is compromised or whether the current recommended daily allowances are unnecessarily high.     

Highly Methylated 6-Hydroxyflavones and Other Flavonoids from Thymus piperella

From the aerial parts of THYMUS PIPERELLA, the free flavone aglycones 5,6-dihydroxy-7,8,4'-trimethoxyflavone ( 1), 5,6-dihydroxy-7,8,3',4'-tetramethoxyflavone ( 2), 5,6-dihydroxy-7,3',4'-trimethoxyflavone ( 3), ladanein ( 4) (5,6-dihydroxy-7,4'-dimethoxy-flavone), 5-hydroxy-7,4'-dimethoxyflavone ( 5) and apigenin ( 6), the flavanones naringenin ( 7) and eriodictyol ( 8), and the flavone glycosides vicenin-2 ( 9), apigenin-7- O-beta- D-glucoside (10) and luteolin-7- O-beta- D-glucoside ( 11), have been isolated and identified. This is the first time that 5,6-dihydroxy-7,8,4'-trimethoxyflavone has been found as a natural product.     

Early knee motion after open and arthroscopic anterior cruciate ligament reconstruction

The hypothesis proposed in this study was that the initiation of active and passive knee motion within 48 hours of major intraarticular knee ligament surgery would not have the deleterious effects of increasing knee effusion, hemarthrosis, periarticular soft tissue edema, and swelling. We conducted a prospective study with randomized assignment of 18 patients into two groups: 9 patients in the "motion" group began 10 hours of daily continuous passive motion (CPM) on the 2nd postoperative day, while the remaining 9 in the "delayed motion" group used a soft hinged knee brace with knee hinges locked at 10 degrees of flexion and entered into the motion program on the 7th postoperative day. All knees were allowed full 0 degrees to 90 degrees of motion except for a total of seven knees with concomitant mensicus repairs and extraarticular reconstructions where 20 degrees to 90 degrees of motion was allowed, limiting the last 20 degrees of knee extension for the first 4 postoperative weeks to protect the repair. In all other respects, the rehabilitation program after surgery was the same for the two groups, including postoperative compression dressings, exercises, and weight-bearing status. Ten of the eighteen patients had acute ACL disruptions and 8 had chronic ACL insufficiencies. There was an even distribution of acute and chronic knee cases and of open and arthroscopic ligament procedures in the early and delayed motion groups. Associated surgery included four meniscus repairs, three medial collateral ligament repairs, and one lateral collateral ligament repair. Special suturing and fixation techniques were used at surgery to maintain the integrity of ligament and meniscus structures, allowing the surgeon to feel safe in subjecting the joint to early postoperative motion. The objective parameters measured were KT-1000 arthrometer measurements, Cybex isokinetic testing, girth measurements at four lower limb locations, range of motion goniometer measurements, postoperative pain medications, and days of hospitalization. Starting intermittent passive motion on the 2nd postoperative day did not increase joint effusion, hemarthrosis, or soft tissue swelling. In both motion groups, postoperative joint effusions were absent after the 14th postoperative day. There was no statistically significant difference in knee extension or flexion limits, pain medication used, or hospital stay in comparing the two knee motion programs. An important finding of this study was the significant decreases in thigh circumference that occurred within the first few weeks of surgery, which progressed despite a closely supervised inpatient and outpatient rehabilitation program.(ABSTRACT TRUNCATED AT 400 WORDS)