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Nanoscale mechanical forces generated by motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces is important to developing motile biomimetic nanodevices powered by biological motors for nanomedicine. Axonemal dynein motors positioned inside the sperm flagellum drive microtubule sliding and give rise to rhythmic beating. This force-generating action pushes the sperm cell through viscous media. Here we report new nanoscale information on how the propulsive force is generated by the sperm flagellum and how this force varies over time. Using a modified atomic force microscope, single-cell recordings reveal discrete approximately 50-ms pulses oscillating with amplitude 9.8 +/- 2.6 nN independent of pulse frequency (3.5-19.5 Hz). The average work carried out by each cell is 4.6 x 10(-16) J per pulse, equivalent to the hydrolysis of approximately 5500 molecules of adenosine triphosphate. The mechanochemical coupling at each active dynein head is approximately 2.2 pN per adenosine triphosphate molecule and approximately 3.9 pN per dynein arm. From the clinical editor: In this paper, nanoscale mechanical forces generated by axonemal dynein motors derived from sperm flagellum are examined and reported. These motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces is important to developing motile biomimetic nanodevices powered by biological motors for nanomedicine., (2010 Elsevier Inc. All rights reserved.)

DNA is packaged within the sperm cell nuclei of many vertebrates and all mammals in a highly condensed state by small basic proteins (protamines). Despite continuous investigation for nearly half a century, the actual packing arrangement of the DNA has remained unresolved. Atomic force and electron microscopy studies described in this report provide evidence that the fundamental packing unit for sperm DNA is a toroidal structure, 900A in outside diameter with a 150A diameter hole, which contains up to 60kb of DNA. Although the results presented here are based primarily upon investigations of mammalian sperm cells, they are expected to be valid for all sperm cells which utilize protamines to package DNA.

When mammalian protamine is dissolved in aqueous buffers at neutral or alkaline pH, both ends of the protein fold inward toward the center of the molecule and form disulfide crosslinks that stabilize several different structures. In the absence of reducing agents, these folded forms of protamine may be visualized and quantitated by gel electrophoresis. Using this technique, we have examined the formation of bull protamine disulfides in solution and describe a variety of factors that affect this process. At pH 8, disulfide-stabilized folded forms of protamine appear within minutes after solubilization of the fully reduced protein. Five different monomers are detected by electrophoresis. Each of these monomers is stabilized by at least one disulfide crosslink and migrates with a distinct mobility, ahead of the fully reduced and extended protein. Under certain conditions, dimers of these folded structures crosslinked by interprotamine disulfides are also formed. The appearance of these disulfide-crosslinked forms of protamine is effected by air oxidation, accelerated at alkaline pH, inhibited upon lowering the pH below pH 7 and eliminated by modifying the protein's cysteine residues. Similar intramolecular disulfides are also produced after the protamine molecule binds to DNA. These results suggest that only those cysteines located within the amino- and carboxyterminal ends of the protein appear to participate in forming intramolecular disulfides in vitro.

Isolated DNA fibers complexed with protamine (the chromosomal protein that packages DNA in mammalian sperm) have been produced by partially decondensing the highly compacted mouse sperm chromatin particle on a glass coverslip. These DNA fibers were then scanned with the atomic force microscope (AFM). While the smallest of the fibers appear in AFM images as ribbon-like structures 250-350 A wide and 10-25 A high, experiments indicate that these images are the result of a convolution of the imaging-tip's shape with the object's actual shape. In such convolutions the height of the object is affected only by the compressibility of the object, while the width is affected in addition by the sharpness of the tip. Images of polyamidoamine particles also appear to show this artifact. We have also deduced the tip's radius of curvature from images of sharp steps and attempt to demonstrate the artifacts associated with a relatively large imaging tip.

The alkylation of histones by the direct-acting carcinogen 7-bromomethylbenz[a]anthracene was demonstrated both in vivo and in vitro. The relative molar reactivity for mouse liver histones in vivo was H3 greater than H1 greater than H2b greater than H4 greater than H2a. The in vitro modification of histone H3 was examined in detail. Amino acid adducts stable to acid hydrolysis were separated after acetylation by reversed-phase high-performance liquid chromatography and characterized using ultraviolet absorbance spectra and synthetic amino acid adduct standards. Three major adducts were observed and tentatively identified as cysteinyl, lysyl and histidinyl adducts of histone H3.