Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase

Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. Thus, the optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. Unless the results of localization using such a series is available, one must be satisfied with adequate but less than optimal results. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was shown to be highly pH dependent. By perfusing the tissue with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This two-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the second fixative solution. The results obtained by increasing the pH of the second solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde maybe obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the second step.     

Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits

Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.     

Development of Photosystem II Complex during Greening of Chlamydomonas reinhardi y-1

The relative content of organized pigment, active centers, and acceptor pools of photosystem II and their interconnection during the development of the photosynthetic membranes of Chlamydomonas reinhardi y-1 have been measured using the fluorescence induction technique. The degree of connectivity and efficiency of the developing system has been assessed also from measurements of maximal rates, quantum yield, and flash yield of 2,6-dichlorophenolindophenol photoreduction using H₂O as the electron donor. The results obtained indicate that the process of membrane development in this organism consists of two phases: an initial phase of reorganization and connection between pre-existing components, and a second phase of actual accumulation of newly formed, complete, and active units. The ratio of active centers to Chl remains practically constant throughout the process while the degree of connectivity between the active center and the plastoquinone pool was doubled during the early phase of the greening. In addition the degree of connectivity between the plastoquinone pool and the rest of the electron transport chain increases as demonstrated by a 10- to 20-fold rise in the quantum yield and a 10-fold rise in the maximal rate and the flash yield. The ratio of light harvesting Chl to active centers remains apparently constant during the second phase of the greening as indicated by light saturation experiments and by the constancy of the apparent photosynthetic unit size. Electron donation from H₂O seems to develop slower than the activity of the rest of the complex as demonstrated by measurements of 2,6-dichlorophenolindophenol photoreduction using 1,5-diphenylcarbazide as the electron donor. The value of all the above parameters which remain constant during the second phase of the greening are comparable to those obtained with membranes of light-grown cells.     

On the thermodynamics of microbial growth processes

In this article, we provide a rigorous thermodynamic analysis of microbial growth process, clarify the role of the generalized degree of reduction concept as it is used in both stoichiometric equations and as a characterizing factor for thermophysical properties, and introduce a classification method to account for errors when using the generalized degree of reduction to estimate the energy and free energy contents of molecules. We maintain the advantages of using the generalized degree of reduction while correcting for the large errors in the principle of energy regularity, especially for small molecules and for nitrogen-source compounds. As a result, we obtain more accurate energy balances (heat loads) and second law constraints, and are able to clarify contradictory statements in the literature as to whether nonphotosynthesis fermentation process can produce oxygen or absorb rather than produce heat. Indeed, the answers to such questions become evident using the classification system introduced here.     

Use of a permanent tissue expander for breast reconstruction

The use of tissue expansion in breast reconstruction is a recognized alternative surgical approach. The second generation of tissue-expanding prostheses is the permanent reverse double-lumen expander. In a series of 100 breast reconstructions using a permanent tissue expander, the clinical benefits of producing a moderate degree of mound ptosis along with observed reduction in pain during expansion are discussed. Additional advantages to the use of the device are the cost savings realized, elimination of a second procedure and a second anesthetic exposure, and a high degree of patient acceptance and satisfaction. This study includes breast reconstruction following mastectomies for malignant and premalignant disease, as demonstrated in 75 delayed and 25 immediate reconstructions, with the longest follow-up being 3 years. Complications of infection (3 percent), significant capsular contracture (4 percent), and implant failure (3 percent) are the most frequent and major complications observed yet. In no instance was breast reconstruction rendered unachievable in the face of these complications. The demonstrated results achieved with permanent tissue-expanding prosthesis, complemented by the obvious benefits and a low rate of significant complications, endorse this method as a viable alternative approach in reconstructive breast surgery.     

The significance of clutch overlap in Great Tits Parus major

We studied the effects of manipulation of the size of first broods in the Great Tit Parus major on the size and breeding success of second clutches and its relation to the degree of clutch overlap. The rearing of first brood fledglings always overlapped with the laying of the second clutch and in most cases also with the incubation period of the latter. The degree of clutch overlap depended on the size of the first brood, being less when the first brood was large. Clutch overlap also increased with season. Mechanisms affecting the timing of laying of second clutches are discussed. A large first brood imposed reproductive costs. It affected the size of the second clutch by causing it to be delayed; second clutch size decreases with season. It affected the post-fledging survival of second brood young as, in this population, this decreases with fledging date. The breeding success of second clutches was, however, not affected by the size of the first brood, but instead by the weight of the female when rearing the first brood.     

Quantitative evaluation of the allometric transformation of of biological structures

Detection and quantification of allometry is a crucial problem in understanding morphological changes, both for systematic and morphogenetic purposes. A section of S.A.M. (Shape Analytical Morphometry) software system was used for this attempt. It consists of the following steps: a) boundary detection; b) starting point detection; c) size normalization; d) extraction of the fundamental shape by Kth order polynomials; e) finding of symmetry evaluator (S.A.E.) by means of a second degree equation. This last procedure gives an arc-chord complex that expresses a vector for allometry where intercept value was for application point, first degree coefficient was for direction and second degree coefficient was for modulus and versus. The main parameters, isometry fraction and allometry fraction may be understood referring them to morphogenetic models.     

Cardiac Function During Exercise in Obese Prepubertal Boys: Effect of Degree of Obesity

The purpose of the study was to evaluate the dynamics of diastolic and systolic function from rest to maximal exercise using conventional echocardiography and tissue Doppler imaging (TDI) in obese prepubertal boys compared to age‐matched lean controls. Eighteen obese (10 with first degree obesity and 8 with second degree obesity according to French curves, BMI: 23.3 ± 1.8 and 29.0 ± 2.0 kg/m², respectively) and 17 lean controls (BMI = 17.6 ± 0.6 kg/m², P < 0.001), aged 10–12 years were recruited. After resting echocardiography, all children performed a maximal exercise test. Regional diastolic and systolic myocardial velocities were acquired at rest and each workload. Stroke volume and cardiac output were calculated. At rest, obese boys had greater left ventricular (LV) diameters and LV mass. Boys in the first degree group showed no diastolic or systolic dysfunction, whereas boys with second degree obesity showed subtle diastolic dysfunction. During exercise, both obese groups showed greater stroke volume and cardiac output. First degree obese boys exhibited greater systolic and diastolic tissue Doppler velocities than controls, whereas second degree obese boys had lower diastolic tissue velocities irrespective of exercise intensity and lower fractional shortening at high exercise intensities than controls. In conclusion, no impairment in diastolic or systolic function is noticed in prepubertal boys with first degree of obesity. Enhanced regional myocardial function response to exercise was also demonstrated in this population, suggesting adaptive compensatory cardiac changes in mild obesity. However, when obesity becomes more severe, impaired global and regional cardiac function at rest and during exercise can be observed.     

Brief communication: Identification reassessment of the isolated tooth Krapina D58 through occlusal fingerprint analysis

High variability in the dentition of Homo can create uncertainties in the correct identification of isolated teeth. For instance, standard tooth identification criteria cannot determine with absolute certainty if an isolated tooth is a second or third maxillary molar. In this contribution, using occlusal fingerprint analysis, we reassess the identification of Krapina D58 (Homo neanderthalensis), which is catalogued as a third maxillary molar. We have hypothesized that the presence/absence of the distal occlusal wear facets can be used to differentiate second from third maxillary molars. The results obtained confirm our hypothesis, showing a significant difference between second and third maxillary molars. In particular we note the complete absence of Facets 7 and 10 in all third molars included in this analysis. The presence of these facets in Krapina D58 eliminates the possibility that it is a third maxillary molar. Consequently it should be reclassified as a second molar. Although this method is limited by the degree of dental wear (i.e., unworn teeth cannot be analyzed) and to individual molars in full occlusion, it can be used for tooth identification when other common criteria are not sufficient to discriminate between second and third maxillary molars. Am J Phys Anthropol 143:306–312, 2010. © 2010 Wiley‐Liss, Inc.     

Assessment of local influence for the analysis of agreement

The concordance correlation coefficient (CCC) and the probability of agreement (PA) are two frequently used measures for evaluating the degree of agreement between measurements generated by two different methods. In this paper, we consider the CCC and the PA using the bivariate normal distribution for modeling the observations obtained by two measurement methods. The main aim of this paper is to develop diagnostic tools for the detection of those observations that are influential on the maximum likelihood estimators of the CCC and the PA using the local influence methodology but not based on the likelihood displacement. Thus, we derive first‐ and second‐order measures considering the case‐weight perturbation scheme. The proposed methodology is illustrated through a Monte Carlo simulation study and using a dataset from a clinical study on transient sleep disorder. Empirical results suggest that under certain circumstances first‐order local influence measures may be more powerful than second‐order measures for the detection of influential observations.     

Comparative investigation of the dynamic conformational properties of human myeloma immunoglobulin G of different subclasses using impulse NMR

The evidence of the intramolecular flexibility of human myeloma immunoglobulin G belonging to the second, third and fourth subclasses has been obtained using the impulse NMR method. It has been found that the degree of intramolecular flexibility for the myeloma immunoglobulin G belonging to the first subclass decreases significantly by cooling from 37 to 10 degrees.     

Method for co-cluster analysis in multichannel single-molecule localisation data

We demonstrate a combined univariate and bivariate Getis and Franklin’s local point pattern analysis method to investigate the co-clustering of membrane proteins in two-dimensional single-molecule localisation data. This method assesses the degree of clustering of each molecule relative to its own species and relative to a second species. Using simulated data, we show that this approach can quantify the degree of cluster overlap in multichannel point patterns. The method is validated using photo-activated localisation microscopy and direct stochastic optical reconstruction microscopy data of the proteins Lck and CD45 at the T cell immunological synapse. Analysing co-clustering in this manner is generalizable to higher numbers of fluorescent species and to three-dimensional or live cell data sets.     

Sorting minor subpopulations of cells: use of fluorescence as the triggering signal

Flow cytometric cell sorting is commonly used to obtain purified subpopulations of cells for use in in vitro and in vivo assays. This can be time-consuming if the subpopulations of interest represent very low percentages of the cell suspension under study. Often the desired subpopulations are identified by two-color immunofluorescence staining. Generally, cell sorting is performed with a flow cytometer configured to trigger on light scatter signals, then sort windows are set based upon the signals from both fluorescent markers. We demonstrate that triggering the cytometer with the fluorescence signal from antibody staining common to both of the desired subpopulations, then sorting the subpopulations based upon staining of a second marker, substantially increases the speed of cell sorting vis-à-vis traditional methods. This is because undesired events are not analysed, allowing an increase in the throughput rate. While desired subpopulations of cells can be obtained by this method, undesired (i.e., nonstaining) cell "contaminants" increase and may require a second sort. The combined time for the initial enrichment sort and a second sort can be less than sorting once using standard methodology. Alternatively, the degree of contamination may be controlled by adjusting the concentration of the cell suspension and by the sample flow rate.     

On the molecular weight of pectic substances and its relation to their gel strengths

A study has been made on the possibility of determining the molecular weight M_w, the second virial coefficient A₂ and the radius of gyration by measuring the light scattering of pectin aqueous solutions. The experimental data were processed on a computer by using an algorithm, accounting for the effect of microgel formations in the pectic solution. A tendency towards a decrease in M_w when decreasing the degree of esterification for a certain type of pectin was established and was confirmed by the change in the intrinsic viscosity [η]. This fact is explained by degradation during deesterification.

The dependence of the gel strength of the pectic preparations on their degree of esterification, molecular weight M_w and purity (p%) was also studied as well as the dependence of the gel strength on the intrinsic viscosity [η], the degree of esterification and the purity p%. The passive multifactor experiment method was used to obtain regression models which were tested for an optimum in the factors influencing the gel strength. The optimal value for the degree of esterification was obtained at 57–58% which confirmed the view that within this range the pectin macromolecules possess maximum conformational flexibility.     

Leukocytic shifts following pancreatic resection with a plasma scalpel and cryodestruction]

Two series of experiments were carried out on dogs. In the first series, blood leukocytes count was studied after resection of the pancreas using plasma scalpel. The resection caused two-phase leukocyte reaction: neutrocytosis (phase I) and leukocytosis involving mainly lymphocytes and monocytes (phase II). During the first 24 hours after the resection, a sharp increase of the leukocyte intoxication index (LII) was observed. The second series of experiments was performed using the combination of plasma scalpel and cryodestruction. The graphic curves denoted a sharp fall both in total leukocytic counts and in the different types of leukocytes which were plotted separately (neutrophils, eosinophils, etc.) This can be explained by the absence of the second (lympho-monocyte) reaction. Only a slight increase in LII occurred. These results reflect lower degree of the protection and adaptation reactions of leukocytes, this being in favor of a combination of plasma scalpel and cryodestruction during the operation.     

Synchronization of ovulation in yaks (Poephagus grunniens L.) using PGF(2alpha) and GnRH

The objective of this study was to test the efficacy of estrus synchronization in yaks using the Ovsynch protocol. To eight non-lactating cycling yaks were administered GnRH analogue followed by PGF(2alpha) analogue treatment 7 days later and further injected with a second injection of same GnRH analogue 2 days after the PGF(2alpha) analogue administration. Ovulation was detected by rectal palpation at 2 h intervals from the initial signs of estrus till ovulation. For LH and progesterone the blood samples were collected at 15 min intervals starting from 1 h prior to the second injection of GnRH analogue until 6 h later and further at 2 h intervals till 2 h after the ovulation. Ovulation was detected in seven out of eight yaks after Ovsynch treatment. The mean time interval from the second GnRH injection to ovulation was 24.8+/-1.95 h with a range of 20-34 h and the mean interval from the LH peak and ovulation was 19.96+/-1.91 h with a range of 14-29 h. The high degree of ovulation synchronization could be attributed to the highly synchronized LH peaks in the treated animals. It was concluded that this estrus synchronization protocol could be applied for fixed time AI in yak.     

Double chip protein arrays using recombinant single-chain Fv antibody fragments

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.     

Pulse NMR study of phase transitions in dipalmitoyl phosphatidylcholine multilayer systems

Pretransition and main transition of aqueous dipalmitoyl phosphatidylcholine (DPPC) dispersions were investigated by pulse NMR. The second moment M2 inter of the proton absorption line shows significant changes at 42 degrees C and about 35 degree C. Over the whole investigated temperature range between 25 and 50 degree C a superposition of at least two distinct second moments assigned to different molecular regions was observed.     

Postpartum reproductive performance in the multiparous mare fed to obesity

Twenty multiparous Quarter Horse mares were assigned to one of two treatment groups at 40 to 75 d of pregnancy. Group 1 was the control group and the mares were fed to maintain a moderate degree of body fat (condition score 5.5 to 7). Group 2 was the obese group and the mares were fed to achieve (prepartum) and then maintain (post partum) an extremely high degree of body fat (condition score 9). Estrous intensity was evaluated using subjective teasing scores ranging from 0 (rejection) to 4 (maximum receptivity). Mares were artificially inseminated beginning with the second postpartum ovulatory cycle; the study was terminated after 63 d of pregnancy. Duration of estrus, maximum teasing score and the number of mares exhibiting overt estrus (teasing score > 2) did not differ between treatment groups during the first and second postpartum ovulatory cycles. The intervals from foaling to first cycle ovulation, foaling to second cycle ovulation, and first to second cycle ovulation were also similar between treatment groups. All mares in both treatment groups conceived and maintained pregnancy. The first cycle conception rate and the number of cycles per conception did not differ between treatment groups. A high degree of body fat produced by overfeeding during gestation did not adversely affect postpartum reproductive performance in the multiparous mare.     

Effects of physico-chemical factors influencing tyramine production by Carnobacterium divergens

Tyramine production by a strain of Carnobacterium divergens was tested in relation to different conditions of pH, temperature, glucose, oxygen availability, potassium nitrate and sodium chloride content, using a combination of a Doehlert and Plackett-Burman experimental design. A second degree polynomial model was chosen to describe tyramine production which was quantified by high performance liquid chromatography. Maximal tyramine production occurred during the stationary phase in acidic conditions obtained by low initial pH (<5) or addition of glucose (0·6%) to the medium. Production was slower at 5°C than at 23°C and 10% sodium chloride inhibited this production. However, the formation of tyramine was not affected by the presence of potassium nitrate or oxygen availability.