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.     

DNA polymerase insertion fidelity. Gel assay for site-specific kinetics

A quantitative assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase alpha holoenzyme complex and M13 primer-template DNA. Km and Vmax values are reported for correct insertion of A and misinsertion of G, C, and T opposite a single template T site. The misinsertion frequencies are 2 X 10(-4) for G-T and 5 X 10(-5) for both C-T and T-T relative to normal A-T base pairs. The dissociation constant of the polymerase-DNA-dNTP complex, as measured by Km, plays a dominant role in determining the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 +/- 0.7 microM), the Km value is 1100-fold greater for misinsertion of G opposite T (4.2 +/- 0.4 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 +/- 4.2 mM). These Km differences indicate that in the enzyme binding site the stability of A-T base pairs is 4.3 kcal/mol greater than G-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax are relatively small. There is only a 4-fold reduction in Vmax for insertion of G opposite T and an 8-fold reduction for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase alpha seems to be governed primarily by a Km discrimination mechanism.     

Nucleotide insertion kinetics opposite abasic lesions in DNA

A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.     

Cell kinetics of gliomas by serial stereotactic biopsy

The potential proliferative activity of glial tumors has been investigate by serial stereotactic biopsies and by "in vitro" 3H-thymidine incorporation procedure (labeling index, LI). The methodology of this combined approach and the preliminary results in 33 patients are reported. Cell kinetic data have been matched with the histological classification (W.H.O.).     

Membrane insertion kinetics of a protein domain in vivo. The bacterioopsin n terminus inserts co-translationally.

The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum bacteriorhodopsin. Strains were constructed that express the bacteriorhodopsin mutants I4C:H(6) and T5C:H(6), which carry a unique Cys in the N-terminal extracellular domain and a polyhistidine tag at the C terminus. Translocation of the N-terminal domain was detected using a membrane-impermeant gel shift reagent to derivatize the Cys residue of nascent radiolabeled molecules. Derivatization was assessed by gel electrophoresis of the fully elongated radiolabeled population. The time required to translocate and fully derivatize the Cys residues of I4C:H(6) and T5C:H(6) is 46 +/- 9 and 61 +/- 6 s, respectively. This is significantly shorter than the elongation times of the proteins, which are 114 +/- 26 and 169 +/- 16 s, respectively. These results establish that translocation of the bacterioopsin N terminus and insertion of the first transmembrane segment occur co-translationally and confirm the use of the assay to monitor the kinetics of polytopic membrane protein insertion in vivo.     

Temperature and light dependent modifications of chlorophyll fluorescence kinetics in spruce needles during winter

Prompt chlorophyll a fluorescence kinetics at room temperature were measured from intact spruce needles. The fluorescence signal was recorded after varying light pretreatments. During the winter, induction curves showed characteristic changes in both the initial peak of fluorescence FV/FP (FP-FO/FP) and the steady state level F_dr (FP-FT/FP). Winter stress induced decreases in both values which showed close correlation to the light and temperature pre-history of the plants. In February changes in fluorescence induction indicative of a restoration of photosynthesis were detected and these corresponded to a rise of temperature above zero in combination with low light levels. In March increasing light intensity combined with chilling temperatures induced again decreases of both values of chlorophyll fluorescence induction suggesting the occurrence of photoinhibition.     

Dissecting the membrane binding and insertion kinetics of a pHLIP peptide

When it is bound to lipid bilayers, the conformation and location of the membrane pH (low) insertion peptide (pHLIP) depend on pH. This unique feature allows us to explicitly measure the kinetics leading to different membrane-bound states of pHLIP using a model membrane and stopped-flow technique. Our results show that the membrane association kinetics of pHLIP are multiexponential and are consistent with a parallel membrane interaction mechanism. Interestingly, our results also show that the overall rate at which the membrane-inserted state is formed is almost identical to that of formation of the surface-bound state, while prebinding slows the rate of peptide insertion.     

A bone biopsy procedure for neonatal pigs

Neonatal pigs were used to develop a surgical biopsy procedure to remove bone tissue from the ilium of small animals, with potential application for infants and small children. While the neonatal pig was under general anaesthesia, a scalpel was used to make a punch incision down to the ilium. Then a Craig Biopsy Trephine was used to remove a core sample of the bone. The samples ranged from 5 to 15 mm in length and 2 to 3 mm in diameter, with an average dry weight of 34.4 mg. The samples were adequate for mineral (calcium and zinc) analysis in our laboratory and may be equally suitable for histological or biochemical analyses. Surgical trauma was minimal, which permitted each pig to be biopsied every 7 days for 5 weeks without adverse consequences.     

Frictional coefficients of multisubunit structures. I. Theory

The theory of Kirkwood for the translational frictional coefficients of structures composed of subunits has been generalized in two ways in order to consider aggregates of nonidentical subunits. One of these generalizations fails when the sizes of subunits are too disparate; the other, derived from a surface shell distribution of frictional elements, is effective over the whole range of relative sizes. It is shown that, in the limit of a continuous surface distribution, a shell model reproduces Stoke's law for a sphere. Comparison is made between the frictional coefficients of spheres, ellipsoids, and rods modeled by finite numbers of subunits and by continuous shells of frictional elements, and those calculated from other theories. Agreement is generally good, though the shell model for prolate ellipsoids of revolution deviates by a few per cent from the Perrin value.     

Recent advances in bone marrow biopsy pathology

The second quarter of 2009 saw steady advances in bone marrow biopsy (BMB) pathology. The following publications are a personal selection of the highlights. Quality issues in diagnostic immunohistochemistry for BMB have largely been ignored in external quality assurance programmes, and this issue is highlighted. In other areas, publications reflecting advances in flow cytometry and aspirate morphology are discussed where translation to the BMB is possible. Classifications undergo constant change, and several publications address the redefinition of the cut off points between malignancy, benign, and normal. Lastly, current scientific research is presented where it is relevant to the understanding of BMB pathobiology.     

Laparoscopy and laparotomy combined with bone marrow biopsy in staging Hodgkin's disease.

The relative merits of laparoscopy with liver and spleen biopsy and staging laparotomy were studied in 91 unselected patients with Hodgkin''s disease. Laparoscopy with liver and spleen biopsy were combined with needle biopsy of the bone marrow and laparotomy was combined with open bone marrow biopsy. In 65 untreated patients six out of seven with liver or marrow disease, or both, were shown to have extranodal lymphomas in these sites by laparoscopy plus needle marrow biopsy. Among 26 patients who had been treated this finding occurred in six out of 10 patients. Spleen biopsies during laparoscopy detected infiltration by lymphoma in 14 out of 37 (38%) patients with diseases spleens. Morbidity was higher after laparotomy than after laparoscopy. Laparoscopy produced abdominal bleeding secondary to splenic biopsy in two patients. All patients with Hodgkin''s disease should be subjected to laparoscopy plus needle marrow biopsy before undergoing laparotomy.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part I

The spontaneous stratification in long-term bone marrow cultures was illustrated and quantified. The cultures were separated into three hematopoietic layers: nonadherent cells in the supernatant medium, lightly adherent cells on top of the stromal layer, and remaining cells buried within the stromal layer. The cells of each layer were subcultured for 10 days in plastic tubes that inhibit the formation of a stromal layer. Daily samplings with absolute and differential cell counts were obtained. We identified three families of cell disappearance curves and cell types: CFU-s, hemocytoblasts, myeloblasts, and promyelocytes (G1, 2); myelocytes (G3); and postmitotic granulocytes (G4). Also, the numbers of mitotic and necrotic cells were determined. The longest half-time of CFU-s was 2.5 days. Lacking stromal support, CFU-s disappeared faster than other differentiated cells. Generally, these cells maintained their numbers for the first week of subcultures, which was attributable to a temporarily maintained balance of cell death and fresh cell production. After more than 7 days, there was a rapid decline of all differentiated cell types.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part II

A mathematical model of mouse granulopoiesis in long-term bone marrow culture was constructed, based on established in vivo cell kinetic parameters. We applied the model to the cell kinetic experiment presented in Part I. Comparing model-predicted cell kinetics with the experimental data led to iterative testing of several hypotheses. In the final model, the cell kinetics of intact tissue culture flasks were reconstructed, using the experimental data from 10 days of tube culture. Among other things, our analysis suggests that the parameters of normal in vivo granulopoiesis apply to bone marrow culture.     

Dynamic mechanism for the serpin loop insertion as revealed by quantitative kinetics

The serpin conformational change by insertion of the reactive center loop into beta-sheet A plays a central role in multiple physiological consequences such as serine proteinase inhibition, latency and serpinopathic polymerization. To study the dynamic mechanism for the loop insertion, a novel kinetic method was established utilizing the ovalbumin mutant R339T/A352R; the loop insertion progressed after the cleavage of P1-P1' (Arg352-Ser353) by trypsin was quenched at pH 8 and 0.5 degrees C, and different conformers were quantified by separation using ion-exchange HPLC. The apparent first-order rate constant k(app) determined for various R339T/A352R derivatives differing in conformational stability was greatly increased by lowering the pH. The pH-dependence of k(app) indicated that the protonation of side-chain(s) with a pK(a) value of around 4.6 is a pre-requisite for the loop insertion. The theoretical rate constant k for the protonated form calculated from k(app) was highly variable, depending on the ovalbumin derivative; structural modifications that give increased mobility to helix F and the sheet-A half (s3A/s2A/s1A) resulted in a striking increase in the loop insertion rate constant k. The k values were determined at different temperatures for all the ovalbumin derivatives, and DeltaH(double dagger) and DeltaS(double dagger) values for the loop insertion reaction were determined according to the transition theory. The formation of the transition state was highly endothermic with minor entropy gain, requiring a DeltaG(double dagger) larger than 18 kcal/mol, which can offset the hydrogen-bond cleavages between s3A and s5A. These results are consistent with the transition state with an opened sheet A and altered orientation of helix F.