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PubMed2001

Controlled release and ocular absorption of tilisolol utilizing ophthalmic insert-incorporated lipophilic prodrugs.

To control ocular drug delivery, the O-butyryl ester prodrug of tilisolol (BUTL) and the O-palmitoyl ester prodrug of tilisolol (PalTL) were incorporated into an ophthalmic insert. The released TL from BUTL inserts and PalTL inserts in pH 7.4 phosphate-buffered saline until 5 h were approximately 25% and 3% of that from TL inserts, respectively. In addition, BUTL was also released from BUTL inserts. However, PalTL was not released from the PalTL insert. The release of drugs from TL inserts and BUTL inserts was little affected by the addition of bovine serum albumin (BSA) in pH 7.4 phosphate-buffered saline. In contrast, the release of drugs from PalTL inserts were enhanced by the addition of BSA. After application of TL, BUTL, and PalTL inserts to the rabbit eye, the aqueous humor concentration of TL was prolonged compared with TL instillation, and the plasma concentration of TL was much lower than that of TL instillation. The ratios of the area under the TL concentration-time curve (AUC) in the aqueous humor to AUC in the plasma (AUC(aqueous)/AUC(plasma)) after application of BUTL until 8 h were 3.1-fold and 3.8-fold higher than those of the TL insert and PalTL insert, respectively.

Absorption↗

PubMed2005

Theoretical and synthetic investigations of carbodiimide insertions into Al-CH3 and Al-N(CH3)2 bonds.

Carbodiimides are known to insert into aluminum-carbon bonds to form four-membered bidentate amidinate chelate rings. Insertions into Al-R and Al-NR'2 (R, R' = alkyl) have been reported in the literature. We have devised a mechanism for these insertions and modeled it using density functional theory (DFT) calculations. The calculated barrier heights for competitive insertions show the insertion into Al-N(CH3)2 goes through a lower barrier than the reaction with Al-CH3 for diisopropyl carbodiimide due to the necessity of forming a pentavalent carbon intermediate in the Al-CH3 case. However, insertion into Al-CH3 has the lower barrier for the reaction with di-tert-butyl carbodiimide because of steric effects, which is consistent with the published experimental results. We have synthesized aluminum amidinates containing two and three acetamidinate rings via insertion of 2 and 3 equiv of diisopropylcarbodiimide into trimethylaluminum (TMA). The crystal structure for [CH3C(N(i)Pr)2]2 AlCH3 is reported. We have found that, although the first insertion is rapid at room temperature, the second and third insertions require refluxing above 70 degrees C. We have calculated the barrier heights for the first and second insertion and have found that this is due to a higher barrier for the migration of the methyl group in the second insertion. This higher barrier is the result of the lack of an exergic precoordination of the carbodiimide to the metal center, which facilitates the first insertion.

Journal Article↗

PubMed2000

Complications and cost associated with parenteral nutrition delivered to hospitalized patients through either subclavian or peripherally-inserted central catheters.

BACKGROUND AND AIMS: Total parenteral nutrition (TPN) is typically delivered through catheters inserted into the superior vena cava (SVC) via a subclavian or internal jugular vein approach. A peripherally-inserted central venous catheter (PICC), utilizing a cephalic or basilic venous approach, may provide a safe alternative to the standard catheter approach and, because non-physician providers can insert the PICC, may introduce a potential cost-savings to health care institutions. We sought to determine if PICC lines are safer and more cost-effective than the standard central venous catheter approach for hospitalized patients who require TPN. METHODS: One hundred and two hospitalized patients (age range, 18-88 years) who required TPN were prospectively randomized to receive therapy via a centrally-inserted subclavian catheter (n=51) or a peripherally-inserted PICC line (n=51). The primary end-point was the development of a complication requiring catheter removal. Other end-points included catheter infection and thrombophlebitis. Cost associated with insertion and maintenance of each catheter was also studied. RESULTS: Complication-free delivery rate (without the need to remove or replace the catheter) was 67% for subclavian catheters and 46% for PICC lines (P<0.05). The overall infection rate was 4.9 per 1000 catheter days and was similar for each catheter type (P=0.68). PICC lines were associated with higher rates of clinically-evident thrombophlebitis (P<0.01), difficult insertion attempts (P<0.05), and malposition on insertion (P<0.05). No catheter complications resulted in significant long-term morbity or mortality. No significant difference was noted between the two catheter types in terms of aborted insertion attempts (P=0.18), dislodgement (P=0.12), or line occlusion (P=0.25). After standardizing costs for each hospital, the direct institutional costs for insertion and maintenance of PICC lines (US$22.32+/-2.74 per day) was greater than that for subclavian lines (US$16.20+/-2.96 per day;P<0.05). CONCLUSION: PICC catheters have higher thrombophlebitis rates and are more difficult to insert into certain patients when compared to the standard subclavian approach for central venous access in hospitalized patients who require TPN. Because of this, PICCs may be less cost-effective than currently believed because of the difficulty in inserting and maintaining the catheter.

Adolescent↗

PubMedDate not supplied

Anatomy and dimensions of rotator cuff insertions.

The purpose of this study was to devise and implement an accurate and reproducible method of measuring the area and dimensions of the rotator cuff tendon insertions and their distance from the articular surface. Twenty fresh-frozen cadaveric upper-extremity specimens were divided into 2 groups of 10. In group 1 the specimens were dissected, leaving only the most distal rotator cuff tendons attached to the humerus. The periphery of the insertion onto the greater tuberosity was marked at 3-mm intervals. The specimens were then mounted onto a custom jig, and the insertion was mapped by a 3-space digitizer. In group 2 the specimens were prepared by removing all tissues except the rotator cuff muscles and tendons and the joint capsule. The interval between the muscles of the rotator cuff was identified and marked. The rotator cuff muscles and tendons were then removed, leaving only the most distal tendons attached to the tuberosities. The periphery of the individual cuff insertions was then marked as in group 1 and mapped in the same fashion. The articular margin also was marked at similar intervals and mapped with the same technique. The area of insertion of the 3 tendons on the greater tuberosity averaged 6.24 cm(2) (SD, 2.04 cm(2)) in group 1. The mean minimum transverse dimension across the cuff insertion occurred in the mid portion of the supraspinatus, with a mean distance of 14.7 mm (SD, 3.22 mm). In group 2 the mean area of insertion of the supraspinatus was 1.55 cm(2) (range, 0.68-2.64 cm(2); SD, 0.66 cm(2)) and the mean area of insertion for the infraspinatus was 1.76 cm(2) (range, 1.23-2.53 cm(2); SD, 0.40 cm(2)). The distance from the articular margin to the most medial rotator cuff fibers was less than 1 mm along the anterior-most 2.1 cm of the cuff insertion onto the greater tuberosity. No correlation could be made between humeral head dimension and the dimensions of the rotator cuff insertions. The mean minimum medial-to-lateral distance across the rotator cuff insertion was sizeable, at 14.7 mm. This represents the minimum possible distance, as the mathematical calculation to determine the dimensions used a perpendicular measurement. The rotator cuff inserts very closely to the articular margin along the anterior 2.1 cm of the greater tuberosity. These anatomic measurements may be useful in evaluating and reattaching the rotator cuff to the humerus.

Aged↗

PubMed1996

Self-catalyzed insertion of proteins into phospholipid membranes.

We present evidence that the mitochondrial channel, VDAC, when reconstituted into a phospholipid membrane, can catalyze the insertion of other VDAC channels. This property called "auto-directed insertion" was first proposed by Zizi et al. (1995) to explain observations on asymmetric VDAC channels. We found that 2 urea or guanidinium chloride (GdmCl) caused a burst of insertions of VDAC channels when added to the same side as VDAC addition. More strikingly, when added to the opposite side they caused a 10-60-fold sustained yet reversible increase in insertion rate. Protein stabilization by sarcosine eliminated the effect of urea and GdmCl on VDAC insertion. Control experiments showed that water flow, ionic strength, osmotic force, phospholipid type, and membrane potential were not involved. Therefore, although both urea and GdmCl affect the properties of phospholipid membranes, it is more likely that these agents act either by changing the structure of the pre-inserted channels, allowing them to be more effective catalysts for VDAC insertion, or by flowing through the channels and acting on nearby VDAC channels inducing them to insert. Either way, insertion must be occurring next to pre-inserted channels. Urea and GdmCl may mimic chaperones by partially unfolding VDAC and keeping it in an insertion-competent state. "Auto-directed insertion" may ensure both correct targeting and orientation of nascent proteins in vivo.

Catalysis↗

PubMedDate not supplied

Pain following controlled cutaneous insertion of needles with different diameters.

BACKGROUND AND AIM: Needle injections are used daily by millions of people around the world for the administration of various drugs (e.g., insulin), venepuncture, and some neurophysiological procedures. The aim of this paper was to study the influence of the outer needle diameter on the pain evoked by controlled needle insertion. METHODS: An automated needle injection system was used to perform a series of insertions where velocity, angle of insertion, and depth of injection were controlled. The frequency of pain following needle insertions (23G, 27G, 30G, 32G) was recorded together with the pain intensity (measured using the visual analogue scale--VAS) and the occurrence of bleeding. RESULTS: The outer needle diameter was positively and significantly correlated to the frequency of the insertion pain; for example, 63% of insertions with 23G needles caused pain, 53% of insertions with 27G and 31% of insertions with the thinnest (32G) needle (p < 0.0001) caused pain. The thickest needle caused most insertions associated with bleeding. Bleeding insertions were approximately 1.3 times more painful (as indicated by VAS scores) than insertions without concomitant bleedings (p = 0.004). CONCLUSION: By decreasing the outer diameter of a needle, the frequency of insertion pain can be reduced and may encourage patients to adhere to demanding injection regimens such as recurrent insulin administration.

Hemorrhage↗

PubMed2001

An ancient retrovirus-like element contains hot spots for SINE insertion.

Vertebrate retrotransposons have been used extensively for phylogenetic analyses and studies of molecular evolution. Information can be obtained from specific inserts either by comparing sequence differences that have accumulated over time in orthologous copies of that insert or by determining the presence or absence of that specific element at a particular site. The presence of specific copies has been deemed to be an essentially homoplasy-free phylogenetic character because the probability of multiple independent insertions into any one site has been believed to be nil. Mys elements are a type of LTR-containing retrotransposon present in Sigmodontine rodents. In this study we have shown that one particular insert, mys-9, is an extremely old insert present in multiple species of the genus Peromyscus. We have found that different copies of this insert show a surprising range of sizes, due primarily to a continuing series of SINE (short interspersed element) insertions into this locus. We have identified two hot spots for SINE insertion within mys-9 and at each hot spot have found that two independent SINE insertions have occurred at identical sites. These results have major repercussions for phylogenetic analyses based on SINE insertions, indicating the need for caution when one concludes that the existence of a SINE at a specific locus in multiple individuals is indicative of common ancestry. Although independent insertions at the same locus may be rare, SINE insertions are not homoplasy-free phylogenetic markers.

Alleles↗

PubMed1980

Quantitative culture of intravenous catheters and other intravascular inserts.

Quantitative cultures were done on 149 intravenous catheters and 40 additional intravascular inserts. Intradermal and intravascular segments of the insert were cultured separately. The inserts were immersed in broth and flushed. The number of colony-forming units (cfu) per insert was estimated by surface culture of serial dilution of the broth. Nonquantitative culture of undiluted broth was also done. Since all inserts associated with bacteremia had at least 10(3) cfu, inserts greater than 10(3) cfu were considered infected. Staphylococcus epidermidis was more likely than more virulent organisms to colonize an insert without causing bacteremia. The inserts in one bacteremic patient were infected from a distant bloodstream focus; however, in the majority of patients, quantitative intradermal cultures suggested that the insertion site was the portal of entry. In bacteremic patients, either a positive quantitative or a nonquantitative culture identified an infected insert. However, only 33% of positive nonquantitative insert cultures from nonbacteremic patients were confirmed by quantitative insert culture.

Catheterization↗

PubMed1991

Tn554 inserts in methicillin-resistant Staphylococcus aureus from Australia and England: comparison with an American methicillin-resistant group.

We have compared methicillin-resistant (Mcr) Staphylococcus aureus isolates from Australia, the UK and the USA with regard to chromosomal inserts of the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon Tn554. The American isolates were known to have a distinctive Tn554 insert, designated insert 6, which was closely associated epidemiologically with the methicillin-resistance phenotype. Southern blots of DNA from Australian and London, UK Mcr isolates were hybridized with a range of probes related to Tn554. The isolates had similar or identical Tn554 inserts, and we consider them to be a single group, designated 'Australondon'. Australondon isolates were compared in detail with a deletion mutant, ANS62, that had lost the methicillin-resistance determinant mec, plus other resistance determinants resident in the mec region of the chromosome, and with an American Mcr isolate containing Tn554 insert 6. The Australondon isolates had three Tn554 inserts. Sequence analysis with the polymerase chain reaction showed that all of these inserts differed from classical Tn554 in that the 3'-terminal residues of the transposons were reverse complements of the usual GATGTA. One of the Australondon inserts, designated 6B, closely resembled Tn554 insert 6 in the sequence of its left flanking chromosomal DNA. This insert was found to abut the deletion from the mec region which results in strain ANS62. We infer that Tn554 insert 6B is part of the mec region of the chromosome in Australondon isolates, supporting the idea that insert 6 of the American isolates is also part of this chromosomal region.

Australia↗

PubMed2005

Speculum 'self-insertion': a pilot study.

AIM: This paper reports phase II of a pilot study that aimed to determine whether self-insertion of a speculum by women undergoing a pap smear would be more comfortable and lead to an improvement in satisfaction and a decrease in anxiety associated with this procedure. BACKGROUND: Research demonstrates that pelvic examinations are considered by most women to be unpleasant and are routinely associated with embarrassment, apprehension, fear and often some level of discomfort and/or pain. DESIGN: The study used quantitative and qualitative data collection techniques. Phase I (described elsewhere) tested the newly developed Speculum Self-Insertion Satisfaction Questionnaire for content validity, internal consistency and clarity. Phase II pilot study tested the technique of speculum self-insertion. Women's general level of anxiety was measured using the State Trait Anxiety Inventory, both before and after they performed the self-insertion procedure. Women's satisfaction and acceptance of the procedure was measured using the Speculum Self-Insertion Satisfaction Questionnaire and explored through the use of qualitative research techniques. PARTICIPANTS: A total of 198 women attending family planning clinics in Perth, Western Australia, between September and December 2003 were invited to participate in the study. One hundred and thirty-three women agreed to self-insert their own speculum. RESULTS: The study demonstrated that speculum self-insertion was acceptable to most women, especially younger women. Nearly 91% of women either agreed or strongly agreed that they were satisfied with the experience of self-insertion and would choose to self-insert the speculum again. This included the women who had not previously had a speculum examination. The quality of specimen collected was not detrimentally affected by speculum self-insertion. CONCLUSIONS: The results of this pilot research, while needing to be replicated in a larger study, demonstrate that offering women the opportunity to self-insert a speculum during a routine pelvic examination is an acceptable, innovative, simple and cost-neutral change in clinical practice that increases women's comfort and satisfaction and potentially makes sexual health screening less threatening to women of all ages. RELEVANCE TO CLINICAL PRACTICE: Speculum self-insertion may encourage women's attendance at clinics for regular screening. Early diagnosis and treatment will result in better health outcomes for women, families and the community at large.

Adolescent↗

PubMed2004

Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus.

Direct insertion of amino acid sequences into the adeno-associated virus type 2 (AAV) capsid open reading frame (cap ORF) is one strategy currently being developed for retargeting this prototypical gene therapy vector. While this approach has successfully resulted in the formation of AAV particles that have expanded or retargeted viral tropism, the inserted sequences have been relatively short, linear receptor binding ligands. Since many receptor-ligand interactions involve nonlinear, conformation-dependent binding domains, we investigated the insertion of full-length peptides into the AAV cap ORF. To minimize disruption of critical VP3 structural domains, we confined the insertions to residue 138 within the VP1-VP2 overlap, which has been shown to be on the surface of the particle following insertion of smaller epitopes. The insertion of coding sequences for the 8-kDa chemokine binding domain of rat fractalkine (CX3CL1), the 18-kDa human hormone leptin, and the 30-kDa green fluorescent protein (GFP) after residue 138 failed to lead to formation of particles due to the loss of VP3 expression. To test the ability to complement these insertions with the missing capsid proteins in trans, we designed a system for producing AAV vectors in which expression of one capsid protein is isolated and combined with the remaining two capsid proteins expressed separately. Such an approach allows for genetic modification of a specific capsid protein across its entire coding sequence leaving the remaining capsid proteins unaffected. An examination of particle formation from the individual components of the system revealed that genome-containing particles formed as long as the VP3 capsid protein was present and demonstrated that the VP2 capsid protein is nonessential for viral infectivity. Viable particles composed of all three capsid proteins were obtained from the capsid complementation groups regardless of which capsid proteins were supplied separately in trans. Significant overexpression of VP2 resulted in the formation of particles with altered capsid protein stoichiometry. The key finding was that by using this system we successfully obtained nearly wild-type levels of recombinant AAV-like particles with large ligands inserted after residue 138 in VP1 and VP2 or in VP2 exclusively. While insertions at residue 138 in VP1 significantly decreased infectivity, insertions at residue 138 that were exclusively in VP2 had a minimal effect on viral assembly or infectivity. Finally, insertion of GFP into VP1 and VP2 resulted in a particle whose trafficking could be temporally monitored by using confocal microscopy. Thus, we have demonstrated a method that can be used to insert large (up to 30-kDa) peptide ligands into the AAV particle. This system allows greater flexibility than current approaches in genetically manipulating the composition of the AAV particle and, in particular, may allow vector retargeting to alternative receptors requiring interaction with full-length conformation-dependent peptide ligands.

Base Sequence↗

PubMed2005

(+)Insert smooth muscle myosin heavy chain (SM-B) isoform expression in human tissues.

Two smooth muscle myosin heavy chain isoforms differ in their amino terminus by the presence [(+)insert] or absence [(-)insert] of a seven-amino acid insert. Animal studies show that the (+)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (-)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (+)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (+)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (+)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (+)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement (nu(max)) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. nu(max) exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (+)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (+)insert isoform could also account for altered contractile properties observed in human pathology.

Amino Acid Sequence↗

PubMed2003

[Effect of the methods of photoactivation and insertion on the hardness of composite resins].

The purpose of this study was to assess the effect of methods of photoactivation and insertion on the Knoop hardness of the Z100 and Alert composite resins. The specimens were confected in cavities measuring 4 x 4 mm. The insertion of material was carried out by means of two methods: single-portion technique and insertion of two 2-mm-thick layers. When inserted in a single portion, the resin was compressed with a static load of 1 kgf on a glass slab recovered with a polyester strip, in order to remove the excess of material. After the removal of the glass slab and polyester strip, the materials were photoactivated by means of continuous light emitted by a XL 3000 unit with a light intensity of 520 mW/cm2 for 40 seconds; double pulse, with light emission of 150 mW/cm2 for 10 seconds, plus 30 seconds with light intensity of 520 mW/cm2 emitted by a XL 3000 unit; and pulsatile light of 520 mW/cm emitted by the Optilux 400 unit, turned on for 2 seconds and off for 2 seconds, during 60 seconds. The two layers of the material submitted to double insertion were photoactivated in the same conditions as the bulk-inserted material, and the excess of material was also removed from the second layer. After storage in a stove at 37 C and 100% relative humidity for 24 hours, the specimens were embedded in polyester resin, trimmed and polished with sandpaper and diamond slurry. Knoop hardness was assessed in 4 depths with a HMV Shimadzu penetrometer under the load of 50 g during 30 seconds. The data submitted to ANOVA and Tukey's test revealed that Z100 presented greater hardness values; double insertion was better than single insertion; the hardness at the surface was smaller than that at the bottom of the specimen, regardless of other factors. For Z100, all activation methods produced smaller hardness values when double insertion was carried out. Meanwhile, for Alert, continuous light promoted statistical similarity between the insertion techniques. For Z100, there was statistical similarity between the activation methods only when the double-insertion technique was employed. There was statistical similarity between the double-pulse and pulsatile-light techniques for Alert, when the single-portion insertion technique was carried out.

Composite Resins↗

PubMed2003

A randomised controlled trial to evaluate the clinical and cost-effectiveness of Hickman line insertions in adult cancer patients by nurses.

OBJECTIVES: To examine the clinical and cost-effectiveness of image-guided Hickman line insertions versus blind Hickman line insertions undertaken by nurses in adult cancer patients. DESIGN: A cost-effectiveness analysis was carried out alongside a randomised controlled trial. SETTING: A large acute cancer centre in Manchester, UK. PARTICIPANTS: Cancer patients due to have a Hickman line insertion who were over 18 years of age and were clinically and physically compliant with specified protocols. INTERVENTIONS: In order to obtain central venous access for the patient, two interventions were investigated: (i) blind insertion of a Hickman line and (ii) image-guided insertion of a Hickman line. Both interventions involved blind venipuncture of the subclavian vein. In the blind arm, the Hickman line was routinely inserted without the use of image guidance at any point in the procedure. Transfer to the interventional X-ray suite and use of image guidance were options immediately available to the operator during the procedure if required. In the image-guided arm, the position of the guidewire was checked before the Hickman line was introduced and later the Hickman line was positioned with the use of X-ray fluoroscopy. MAIN OUTCOME MEASURES: The primary clinical outcome measure was catheter-tip misplacement and this was expected to be higher in the blind arm. When comparing the skill level of the trainer and the trainees, pneumothorax was the primary clinical outcome measure. Other outcomes measures included arterial puncture, haematoma, infection, failed insertion and assistance from other healthcare professionals. RESULTS: No statistically significant difference was found between the mean cost per patient in the two arms of the trial. The only statistically significant difference in clinical outcomes was the frequency of catheter-tip misplacement, which was higher in the blind arm of the trial. At very low costs, the image-guided approach dominates the blind approach as fewer costs and greater benefits are incurred. It is evident that nurses previously inexperienced in the procedure can be trained to insert Hickman lines successfully both at the bedside and under image guidance within a 3-month period. CONCLUSIONS: This report indicates that nurse insertion of Hickman lines in the majority of adult cancer patients is both safe and effective. However, there are a select group of patients for whom image-guided insertion may be preferred. The results reveal that skills and expertise can be transferred from trainer to trainee through a relatively short, but intensive, training course. It is also evident that patients support nurse insertion. Further research is suggested to compare the safety and efficacy of nurse versus doctor insertions in particular subgroups of patients and also to assess the quantity and quality of current service provision in order to inform NHS decision-making in this area.

Adult↗

PubMed1991

Investigation of early surface delamination observed in retrieved heat-pressed tibial inserts.

The objective was to examine possible reasons for delamination observed in tibial inserts of the porous-coated anatomic (PCA) knee replacement. To date, 33 PCA inserts have been forwarded to the authors' labs. Of these 33, 52% showed severe delamination within four years of implantation. Visual, structural, and mechanical analyses were conducted and data compared on the heat-pressed PCA type and the common machined inserts. Twenty inserts of the two different types were examined. Visual data using polarized light microscopy showed the presence of a surface layer separated from the middle region of the heat-pressed inserts by a line of demarcation 250-580 microns beneath the articulating surface. This anomaly was not observed in machined inserts. Structural analysis showed the new heat-pressed inserts had increased crystallinity in the surface layer when compared to new machined inserts. The retrieved heat-pressed inserts showed increased crystallinity in the surface and middle regions. There was a slight increase in surface crystallinity in the retrieved machined inserts. Microhardness data showed that there was an increased hardness associated with the crystallinity seen on the heat-pressed inserts. Orthopedic surgeons should be aware of early delamination and surface failure in heat-pressed inserts.

Hardness↗

PubMedDate not supplied

[Cloning and study of inserted sequences of gene 28S in Drosophila melanogaster ribosomal RNA].

Cloning of fragments of ribosomal genes containing insertions in the 28S RNA gene has been reported earlier. Subcloning of DNA fragments corresponding to insertion sequences and their hybridization with DNA, RNA and polytene chromosomes from different flies is described. Type 1 insertions (containing BamI sites) are highly heterogeneous in length and sequence even in homozygotes. Type 2 insertions (with EcoRI sites) are rather homogeneous. Two types of insertions are represented in the D. melanogaster genome by 50 and 30 copies, respectively. Restriction fragments with insertions significantly differ in DNA from embryos and larvae. D. simulans and D. virilis also contain the sequences of both types of insertions, though in fewer number of copies. Type 1 insertions seem to be poorly transcribed, and type 2 insertions are not transcribed at all. Among 2000 recombinant clones screened a number of DI plasmids hybridizing to isolated insertions were obtained. Six of them were mapped with restriction endonucleases and hybridized with insertion fragments. rRNA and polytene chromosomes. All of these DI plasmids hybridize with the nucleoli, one with the chromocenter and one with the 79F 3L site. In LI9, not coding for rRNA, the sequences, corresponding to two types on insertions are located only a few kilobases apart. D17a does not encode for rRNA, but hybridizes in situ only with the nucleoli.

Animals↗

PubMed1994

Accommodation of amino acid insertions in an alpha-helix of T4 lysozyme. Structural and thermodynamic analysis.

One to four alanines were inserted by site-directed mutagenesis at three different locations within the alpha-helix comprising residues 39 to 50 in bacteriophage T4 lysozyme. All insertion mutants were correctly folded and catalytically active although the insertions led to a thermal destabilization by 1.1 to 4.2 kcal/mol when compared to wild-type. Variants that restored part of the loss in stability associated with the initial alanine insertions could be found by randomizing the inserted amino acids. In selected cases, directed mutagenesis of adjacent residues was also used to regain stability. Structural information obtained from X-ray crystallography and/or 2D-NMR for 10 different variants showed two distinct ways in which the protein responded to the amino acid insertions: (1) The inserted amino acids were incorporated into the helix by replacing preceding wild-type amino acids and causing a shift in register towards the N terminus. As a consequence, wild-type amino acids were translocated from the helix into the preceding loop. (2) Insertions caused a "looping out" within the alpha-helix. In this case the perturbation was confined to a minimal region in the immediate vicinity of the insertion. No change in the length of the helix was detected in either case. The structural response appears to be determined by the maintenance of the hydrophobic interface between the helix and the rest of the protein. This interface remains essentially intact in all variant structures. The results exemplify the plasticity and the adaptability of the protein structure which allows the incorporation of additional amino acids into a secondary structure element without large structural perturbations, as long as vital internal interactions are preserved. They also suggest that loops in proteins related by evolution can vary in length not only because of insertions within the loops themselves but also as a consequence of insertions within neighboring secondary structure elements.

Alanine↗

PubMed1999

Epitope insertion into variable loops of HIV-1 gp120 as a potential means to improve immunogenicity of viral envelope protein.

We report on the properties of a set of HIV-1 IIIB Env mutants carrying a linear gp41 epitope insertion (LLELDKWASL) in the V1, V2, V3 or V4 variable loop. Insertion of the epitope, which is defined by the HIV-1 neutralizing MAb 2F5, was well tolerated in the V1, V2 and V4 loops, as these mutants were properly expressed, retained reactivity to conformation-dependent monoclonal antibodies and exhibited patterns similar to the parental Env molecule. However, insertion of this epitope in the V3 loop was associated with drastically reduced protein expression. Relative to parental Env molecule, the V1, V2 and V4 insertion mutants demonstrated significantly increased binding to mAb 2F5 in vitro. To evaluate immunogenicity, mice and guinea pigs were immunized with plasmid expression vectors for the mutant proteins. For both mice and guinea pigs, all four mutants elicited anti-gp120 antibody responses. In mice the V1 and V3 insertion mutants, but neither the V2 or V4 insertion mutant nor the parental env, elicited significant titers against the epitope peptide, whereas in guinea pigs, V2 insertion mutant was most effective in eliciting anti-2F5 peptide antibody responses. While original V2 2F5 insertion mutant failed to elicit anti-2F5 peptide responses in mice, studies with 14 additional V2 insertion mutants revealed several insertion sites at which the epitope was able to induce epitope-specific antibody responses. This indicates that the precise position at which the epitope insertion takes place dictates the ability of the mutant to induce the epitope-specific antibody responses. When tested for virus neutralization activity, the guinea pig sera that contain high titers of anti-2F5 peptide antibody failed to enhance the virus neutralizing activity, suggesting that the configuration of 2F5 epitope plays a critical role in inducing neutralizing antibody responses. The results from this study may have potential implications with respect to modification of the HIV-1 Env molecule for the purpose of improving HIV-1 Env immunogenicity.

AIDS Vaccines↗