Operating conditions for preparative successive solutional fractionation, 1. Effect of initial concentration and fraction size

Operating conditions, such as initial concentration, v⁰_p, fraction size, Q, in preparative successive solutional fractionation (SSF) based on solubility are studied in comparison with successive precipitational fractionation (SPF), on various polymers having Schulz-Zimm and Wesslau distributions. The partition coefficient σ is larger if SSF is performed on more dilute solutions. The volume ratio, R, of the polymer-lean phase to the polymerrich phase in SPF is always large as compared with SSF. For the middle fractions obtained by SSF or SPF the content of both higher and lower degree of polymerization (X) regions in the molecular weight distribution decreases as v⁰_p decreases. The v⁰_p dependence of X?_w/X?_n in the fraction is remarkable in SPF as compared to SSF. SSF at a v⁰_p lower than 0,1% is not effective for the preparative method and an unlimited decrease in X?_w/X?_n (till 1) of the fraction can not be expected. The fractionation efficiency does not reduce to zero even if v⁰_p is in vicinity to the critical concentration. Neither X?_w/X?_n nor the standard deviation σ' in the fractions obtained by SSF exhibit a maximum at a specific v⁰_p. In contrast to SPF, in SSF X?_w/X?_n in the fractions decreases without limit (to 1) with increasing total number of fractions, n, compared at a given X?_w. Actually n = 25–30 is necessary for preparation purposes. With a decrease in Q the X?_w/X?_n vs. v⁰_p relations shift to lower X?_w/X?_n and the total profile of the relations is not essentially influenced by Q except for the higher v⁰_p region.     

Operating conditions for successive precipitational fractionation of macromolecules, 2. Analytical fractionation

Operating conditions, including the initial concentration, the total number of fractions, n, and some refractionation procedures, in the successive precipitational fractionation (SPF) are investigated as well as the reverse-order fractionation phenomena in the case of analytical fractionation. SPF is ineffective for determining of the molecular weight distribution of the original polymer if its polydispersity is very small. The SCHULZ plot, obtained from the data for the total number of fractions, n = 4, is practically indistinguishable from the corresponding plot for n = 20 to 30. In contrast to this, the KAMIDE plot approaches the true distribution with increasing n. The breadth in the molecular weight distributions remains effectively constant for n ≥ 10, compared with the same weight-average molecular weight. The reverse-order fractionation hardly occurs, from the theoretical point of view, provided that the polymer-rich and polymer-lean phases are in thermodynamic equilibrium. The ratio of the weight- to number-average degree of polymerization, X?_w/X?_n, in the fractions obtained by refractionation procedures does not approach an asymptotic value even if the refractionation is repeated five times. The refractionation with the fraction size, ρ = 1/2 is found to be very effective. For a given pair of polymer and a solvent, the minimum value of X?_w/X?_n attainable from a solution of a given initial concentration, by SPF, can be calculated by a computer simulation technique.     

Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen

Melanoma antigen recognized by T cell 1 (MART-1) is regarded as a candidate peptide for vaccination against malignant melanoma, and it is of importance to develop strategies to improve the vaccine-elicited T-cell activation towards MART-1. T-cell activation is, among other determinants, dependent on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared to non-proteasomal targeting of the epitope to increase its cell-surface presentation. Furthermore, we explored the potential of incorporating multiple minigenes instead of one to increase cell-surface presentation. We show that both proteasomal targeting and repetition of the minigene increase cell-surface presentation of the epitope and propose both these approaches as potential strategies in DNA vaccines to increase MART-1-specific T-cell activation.     

A modified procedure for the identification of anaerobic bacteria by high performance liquid chromatography--quantitative analysis of short-chain fatty acids.

We have developed a new rapid method for analysing volatile and non-volatile short-chain fatty acids using high-performance liquid chromatography. Within 50 min, 22 fatty acids in a standard mixture could be detected in a single chromatographic run. The fatty acids released by anaerobic bacteria in the culture media were ether-extracted and analysed with an Aminex HPX-87H column. Using a microprocessor-controlled chromatography unit, a quantitative analysis of the fatty acids produced in bacterial cultures was possible. Resolution, rapidity and sensitivity were improved as compared to previous methods by using an eluent of 5% acetonitrile in 0.01 N H2SO4 and changing the column temperature to 35 degrees C.     

Use of a modified procedure for treating small amounts of red blood cells with 2-aminoethylisothiouronium bromide

Kell null red blood cell samples can be prepared using 2-aminoethylisothiouronium bromide (AET). This article describes a modification whereby only three drops of a 5% red blood cell suspension may be AET treated. This procedure has been used routinely in the authors' laboratory for more than a year. One patient's serum contained anti-Kpb plus anti-C and anti-D. Tests with panel cells pretreated with AET made it possible to identify underlying Rh antibodies without using a panel of genetic Kpb negative red blood cells. Of 24 red blood cell eluates from patient sample with warm autoantibodies, one had specificity within the Kell blood group system. This small volume AET-treatment method is a quick screen for the differentiation of a Kell-related specificity from a non-Kell specificity of both warm autoantibodies and alloantibodies to high-incidence antigens.     

A modified mixed haemadsorption procedure for the detection and localisation of sperm antigens.

A mixed haemadsorption procedure for the detection and localisation of spermatozoal antigens is described. Sperm are immobilised by attaching them to the bases of wells on plastic plates using poly-L-lysine and antisera and sensitised red cells are then added to these wells. The technique uses simple reagents and allows sperm/red cell rosettes to be kept as permanent records.     

A method for the preparation of Vicia villosa lectin and a Rosette procedure for fractionation of lectin-binding lymphocytes

We describe a simple procedure for isolation of Vicia villosa lectin with binding specificity for N-acetyl-D-galactosamine >D-galactose. Also, a rosette assay using V. villosa lectin coupled-SRBC is described which enables us to both visualize lectin binding cells and to fractionate lymphocytes from in vitro primary mixed lymphocyte cultures on the basis of ability to bind V. villosa lectin.     

Continuous fractionation and solution properties of PVC, 2. Scrutiny of solvents,intrinsic viscosity

Investigations with ca. 100 organic liquids in a temperature range from ?20°C to 90°C result in a survey of the solubility behaviour of PVC. The experimental results were compared with the predictions of the solubility parameter theory: Accepting δ = 9,75 (cal/cm³)^1/2 as the solubility parameter for PVC, all solvents (except ketones in which PVC is soluble independent of their solubility parameters) are located within the range of δ_PVC ± 0,75. Only three of the 100 liquids examined turned out to be (endothermal) theta-solvents, namely the aromatic solvents o-xylene, phenetole and dimethyl phthalate. The theta-temperatures in o-xylene and phenetole were determined viscometrically by means of the temperature dependence of the Mark-Houwink exponent to be 84 and 88°C, respectively. Viscosity measurements were also performed in the temperature range of 25 to 60°C with solutions of PVC 75 000 in 10 thermodynamically good solvents used for rheological investigations. In all cases the intrinsic viscosities decrease slightly with increasing temperature, i.e., all solutions are weakly exothermal. The binary cluster integral, β, was determined (taking the monomeric unit as the statistical segment) and the polar contribution, β_e, was calculated from Yamakawa's theory. The correlations between the non-polar part of (β_n = β – β_e) and the solubility parameter of the solvent, found by Utracki, were tested and found to be valid for the present systems, too.     

Applications of a modified Cortipac procedure for the estimation of urinary free cortisol in various clinical situations.

A modification of the Cortipac procedure (Radiochemical Centre, Amersham) for the estimation of urinary free cortisol was found to be of value in the diagnosis of Cushing's syndrome and in the differentiation of simple obesity from that due to Cushing's syndrome. The effect of renal failure and late pregnancy on the urinary free cortisol was also studied. The results were expressed both in terms of the urinary free cortisol and as a urinary free cortisol:creatinine ratio.     

光电显示颈内静脉穿刺标本的制作与应用

目的:为改良Elmslie-Trillat术治疗髌骨不稳定提供生物力学依据并探讨临床疗效.方法:实验组新鲜冰冻膝关节完整标本制成髌骨不稳定模型,然后采用改良Elmslie-Trillat术矫正,在MTS试验机上,通过髌骨中心外侧施加相应的载荷,传感器记求髌骨移位距离.临床组关节镜下髌外侧支持带松解及改良Elmslie-Trillat术治疗复发性髌骨脱位20例21膝临床分析.结果:实验组改良Elmslie-Trillat术后标本与膝关节完整标本生物力学差异无统计学意义(P>0.05).临床组术后随访平均31个月(14～52个月),未见髌骨脱位复发.Lysholm膝关节功能评分手术前后分别为(62.3±5.1)分和(90.1±5.3)分(P<0.01),CT检查显示股骨髁滑车凹中心与胫骨结节水平距离(TA-GT)从术前(17.80±1.4)mm改善为(10.60±1.6)mm(P<0.01).结论:根据生物力学原则,改良Elmslie-Trillat术能有效治疗髌骨不稳定,恢复膝关节功能.     

SJ-GC, a modified complete medium for growth of Neisseria gonorrhoeae.

A broth medium that supports growth of Neisseria gonorrhoeae to high densities with minimal lysis is described. With most gonococcal strains, inocula of 2 x 10(6) colony-forming units per ml yielded greater than 109 colony-forming units per ml after 8 h of incubation. Scale-up cultures produced 5 to 12 g (wet weight) of cells per liter.     

Low dose-response for 2-deoxy-D-glucose-induced feeding, and the involvement of peripheral factors.

Latencies to feed and meal sizes were observed in recently satiated rats injected intraperitoneally with an isotonic solution of 2-deoxyglucose, using 3-0-methyl glucose for the control injection. The feeding response latency shortened monotonically with dose from about 0.3 mmole/kg (50 mg/kg) to 0.5-0.6 mmole/kg, with an extended plateau up to dehilitating doses. At modest doses, the latency to feed was shorter in adrenal demedullated rats than in intact rats, suggesting that the hyperglycemic response to 2DG partially inhibits the feeding response. Food-deprived rats took larger meals following 2DG injection, if there had been recent rapid intestinal absorption of glucose. It is suggested that a major action of 2DG is to block the satiating consequences of the process of absorption, possibly at hepatic receptors.     

Isolation of a DNA probe for identification of Mycobacterium kansasii, including the genetic subgroup.

In order to develop a DNA-based assay to identify all Mycobacterium kansasii clinical isolates, a specific DNA probe was isolated in plasmid p6123. A total of 145 M. kansasii clinical isolates were collected from several countries and were examined with three probes by DNA hybridization. Of the 145 isolates, 115 (79%) were positive with the previously described probe pMK1-9 (Z. H. Huang, B. C. Ross, and B. Dwyer, J. Clin. Microbiol. 29:2125-2129, 1991), 129 (88%) were positive with the commercial Accu-probe assay (Gen-Probe), and 145 (100%) were positive with the p6123 probe. Southern blot analysis of EcoRI-digested M. kansasii chromosomal DNA with p6123 revealed that all Accu-probe-positive M. kansasii strains exhibited a 3-kb fragment, whereas all Accu-probe-negative M. kansasii strains displayed DNA fragments of variable molecular sizes. These results indicate that, unlike the previously described probes for M. kansasii, the fragment cloned into p6123 identified all 145 biochemically typical strains tested and provides an ideal target for future DNA-based speciation assays.     

Isolation of a temperature-sensitive dengue-2 virus under conditions suitable for vaccine development.

Dengue virus, type 2, in viremic human sera and after passage in cell cultures produces mixtures of small and large plaques when assayed in LLC-MK2 cells. Clones of dengue virus type 2 obtained by plaque selection in primary green monkey kidney cell cultures were tested for temperature sensitivity in vitro and for virulence by intracerebral inoculation of suckling mice. Sublines of a small-plaque clone were found to have lower nonpermissive temperatures than the parent virus by both plaque formation and release of infectious virus into the culture media. Small-plaque sublines were significantly less virulent in suckling mice than was the parent virus. Sublines from a large-plaque clone were not temperature sensitive and closely resembled parent virus mixed-plaque morphology. When small-plaque sublines were serially passaged using undiluted inocula, reversion occurred as evidenced by the appearance of large plaques and return of mouse virulence. Small-plaque virus could be maintained through several serial passages without reversion by using low-input inocula. Desirable passage history as well as temperature-sensitive and attentuation characteristics of the S-1 small-plaque subline make it appear suitable as a vaccine candidate virus.     

Bone marrow transplantation in Polish conditions. A modified method of marrow collection and preparation for transplantation

Marrow is collected from posterior iliac crests using specially developed "Wiktor's" needles and from other sites using commercially available sternal needles into heparinized syringes. Then it is directly injected into collecting flasks through specially designed bone marrow filters. This assures single cell nature of marrow cell suspension which then may be either directly infused into the recipient or processed for red cell or T cell depletion. During collection marrow cell suspension is mixed using magnetic stirrers. The described method allows collection of sufficient number of marrow cells for engraftment at the expense of acceptable risk for the donor. Data for 10 consecutive marrow collections are reported.     

The use of a modified administrative procedure (MAP) for the bender-gestalt test with schizophrenic patients and normals

Advocated a modified procedure for administering the Bender Gestalt Test that involves administering the BG in a conventional way and, after a brief intervening period, a second administration with specific instructions to copy the BG designs exactly. Three subtypes of schizophrenia and a normal control group were studied: 25 paranoids, 25 chronic undifferentiated, 25 schizoaffectives, and 25 controls. Each schizophrenic subgroup demonstrated significant improvement in performance on the BG when the modified administrative procedure was employed. Some implications of the findings are discussed.     

A further case of a new syndrome including midface retraction, hypertrichosis, and skeletal anomalies.

The clinical and radiological features of a male child with multiple malformations are described. He appears to be an example of a new syndrome recently described by Schinzel and Giedion (1978).     

Clinical evaluation of a simple, rapid procedure for the presumptive identification of anaerobic bacteria.

A simple, rapid procedure for the presumptive identification of anaerobic bacteria has been evaluated. Two hundred and thirty-five clinical isolates were identified using gas-liquid chromatography and 3-ml volumes of a few selected test media. These test media were stored aerobically and incubated in GasPak anaerobic jars. The average incubation time was 39 h. This procedure, when compared to the results of our standard identification procedure, correctly identified 98% of the isolates to the genus level, 83% to the species level, and 83% of Bacteroides fragilis and Bacteroides melaninogenicus to the subspecies level. Fifty-three of the isolates were also identified by using 0.5-ml volumes of test media stored, inoculated, and incubated in an anaerobic glove box. The 3-ml-and the 0.5-ml-volume procedures correctly identified comparable percentages of the 53 isolates.     

Genetics of experimental lupus nephritis: non-H-2 factors determine susceptibility for renal involvement in murine chronic graft-versus-host disease.

Chronic graft-versus-host disease (GvHD) was induced in (C57BL/10 x DBA/2)F1 and (B10.S x DBA/2)F1 hybrids by injection of DBA/2 lymphocytes. All of the animals developed GvHD. Renal disease and proteinuria occurred in all of the (C57BL/10 x DBA/2)F1 hybrids, but only in 54% of the (B10.S x DBA/2)F1. The type of renal lesion was similar in all diseased animals of both strains, i.e., immune complex glomerulonephritis (ICGN) with deposition of antibodies and complement in glomeruli. To find out whether H-2 haplotype or other factors, such as non-H-2 linked genes, determine the susceptibility for renal involvement in GvHD, we produced (B10 x B10.S)F1 x DBA/2 mice, determined their H-2 genotype serologically, and separated them into H-2b/d and H-2s/d groups. These two groups did not differ with respect to susceptibility to renal disease in the course of GvHD, which indicates that H-2 is not the decisive genetic factor. We conclude that factors not linked with H-2 exert a major influence on susceptibility to GvHD-related renal disease in these mice.