A heterobifunctional crosslinker was synthesized from a diamine derivative of poly(ethylene glycol) (PEG, average molecular weight 800 Da), with the functional groups 2-(pyridyldithio)propionyl (PDP) and N-hydroxysuccinimide ester (NHS). The crosslinker can be used for linkage of two different proteins for which a suitable protocol is presented, explified by crosslinking of two antibodies with 50% yield. In a second application the crosslinker is used to generate immunoliposomes. The NHS group was reacted with an aminolipid for liposome anchorage, and antibodies were bound to the PDP group via disulfide bonds. Loading of liposomes with antibodies was easily adjustable, even down to only a few per liposome. This crosslinker with its particular length appears especially suited for the flexible anchorage of biomembranes, opening new perspectives in membrane research as discussed.

Amines↗

PubMedDate not supplied

Basic studies on heterobifunctional biotin-PEG conjugates with a 3-(4-pyridyldithio)propionyl marker on the second terminus.

Heterobifunctional poly(ethylene glycol) (PEG) derivatives with a biotin terminus have been synthesized and characterized with respect to avidin binding. Unambiguous measurement of biotinyl and pyridyldithiopropionyl end groups was established by selecting suitable assays and introducing necessary modifications. Functional studies on the binding of biotin-PEG conjugates to avidin tetramers revealed much similarity to known biotin-spacer-peptide conjugates with 7-27 atom spacers: dissociation kinetics of the initially formed 4:1 complexes were multiexponential, the complex with 2 ligands per avidin dissociating rather slowly with half-times of approximately 2 days at 25 degrees C. The observed stability of 3:1 and 2:1 complexes with avidin is particularly significant since it allows exploitation of the additional advantages of PEG spacers, i.e. reduced steric strain in biotin-avidin-biotin bridges, reduced nonspecific adsorption of biotinylated probes and markers, and, especially, uncomparable fluorescence intensities of biotin-PEG-fluorophore conjugates as is demonstrated in the accompanying study (second of three papers in this issue).

Avidin↗

PubMedDate not supplied

Biotin-fluorophore conjugates with poly(ethylene glycol) spacers retain intense fluorescence after binding to avidin and streptavidin.

Conventional biotin-fluorophore conjugates with approximately 14 atom spacers lose most of their fluorescence when binding to avidin or streptavidin, as is demonstrated in the present study. This explains the unusual fact that only biotinylated marker enzymes, but not fluorescent biotins, are regularly used in bioanalytic assays. Novel biotin-spacer-fluorophore conjugates are presented that retain intense fluorescence when binding to avidin or streptavidin. Preservation of fluorescence depends upon the use of poly(ethylene glycol) (PEG) spacers, which are shown not to interfere with biotin function. The observed absence of nonspecific interactions may also be attributed to the PEG chain. These novel fluorescent biotins are expected to be excellent new tools in fluorescence microscopy and related techniques.

Avidin↗

PubMedDate not supplied

Porin channel triplets merge into single outlets in Escherichia coli outer membranes.

Previous observations on the structural and functional properties of porin, the matrix protein of Escherichia coli, have indicated that the channel-forming trimers span the outer membranes of the bacterial cell, forming a molecular sieve. By using electron microscopy and image reconstruction, we demonstrate here that three channels on the outer surface of the cell merge into a single channel at the periplasmic face. Conductance measurements using conditions under which single activated triplets could be observed led us to conclude that the three individual consecutive closing steps reflect three channels within a single trimeric unit. Statistical analysis of conductance levels revealed that the first relaxation step is distinctly smaller than the two subsequent channel closings. This functional observation can be explained if the channels of porin trimers coalesce.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

[Neurologic and psychosocial disorders in children with brain tumors].

We studied 15 tumor-free patients, that had already finished treatment at the university-hospital Essen. Beside from a neurological check-up our complete assessment included the following tests: CMM (Columbia Mental Maturity Scale), BM and CM (Raven Test), PPVT (Peabody Picture Vocabulary Test), BA (Following Directions), KP (Dotting), d2, HAWIK-Test (Mathematical part), LOS (Lincoln Oseretzky Motor Development Scale) and GFT (Göttinger Formreproduktionstest). A half structured interview of parents was performed, too. All, but two patients, had neurological disorders of different degrees. All these 13 patients had signs of ataxia. For the intelligence- and development-tests (CMM, BA, BM, CM and PPVT) the results for all patients were better than the 50th percentile, for the KP and LOS-test worse (40-47%). A diminished ability for concentration and slowness shows the d2-reached only a rate of 22 percent. The results of the HAWIK-test shows a test-age-equivalent, which is 3,4 years above the actual age. From the results of the GFT you can conclude, that these disorders are caused by brain damage. Parents mentioned during the interviews abnormalities in behaviour, interaction-problems between siblings and inadequate emotional reactions. For all these reasons we want to point out, that for an overall assessment of these patients and there families a well-planed therapeutical procedure including rehabilitive measures is necessary for diminishing the described disturbances as far as possible.

Adaptation, Psychological↗

PubMedDate not supplied

Single molecule microscopy of biomembranes (review).

Recent advances in the development of new microscopy techniques with a sensitivity of a single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are analysed here in terms of their applicability to the in vivo visualization of cellular processes on the molecular scale, in particular of processes in cell membranes. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultrasensitive methodology as a general tool to study local processes and heterogeneities in living cells.

Animals↗

PubMedDate not supplied

[Epidemiological pattern of lymphatic filariasis in children living in endemic areas].

OBJECTIVE: Lymphatic filariasis still represents a major public health problem in the city of Recife. In spite of the fact that previous surveys had already shown high frequency of microfilaraemia in pediatric population, the prevalence of filarial disease and the microfilaraemic pattern of this group were unknown. This paper describes the clinical-epidemiological pattern of filariasis in children and adolescents living in two highly endemic areas of Recife. METHODS: The parasitological survey was done through a census carried out between December 1990 and July 1991. Thick drop technique (45 micro l) was performed on a total of 1,464 children and adolescents between the ages of 5 and 14, of whom 967 were submitted to clinical examination. Positive cases had their blood recollected (60 micro l) to measure the microfilaraemic density. RESULTS: The microfilaraemia prevalence was 6.4 %. In the age groups of 5 to 9 and 10 to 14 a microfilaraemia prevalence of respectively 4.6% and 8.3% was observed. The microfilaraemic density varied from 3 to 864 microfilariae per 60 microl of blood, there having been no statistically significant difference between the sexes and age groups (p<0.05). 6 cases (0.6 %) of acute filarial disease and 11 of chronic filarial disease (1.1%) were identified, hydrocele being the principal manifestation found. Lymphadenopathy was found in 22% of the children, statistical association with microfilaraemia being observed (p<0.001). CONCLUSIONS: The results of the parasitological survey show the strong presence of children in the contingent of microfilaraemic individuals, indicating an early and intense exposure to filariasis in the population studied.

English Abstract↗