Molecular specific and cell selective cytotoxicity induced by a novel synthetic HLA-DR antibody mimic for lymphoma and leukemia.

Like rituximab, monoclonal antibodies reactive with human leukocyte antigen have potent antilymphoma activity. However, size limits their vascular and tissue penetration. To mimic monoclonal antibody binding, nanomolecules have been synthesized, shown specific for the beta subunit of HLA-DR10, and selective for cells expressing this protein. Selective high affinity ligands (SHALs) containing the 3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid (Ct) ligand residualized and had antilymphoma activity against expressing cells. Herein, we show the extraordinary potency in mice with human lymphoma xenografts of a tridentate SHAL containing this ligand. After titrating antilymphoma activity in cell culture, a randomized preclinical study of a tridentate SHAL containing the Ct ligand was conducted in mice with established and aggressive human lymphoma xenografts. Mice having HLA-DR10 expressing Raji B- or Jurkat's T-lymphoma xenografts were randomly assigned to receive either treatment with SHAL at a dose of 100 ng i.p. weekly for 3 consecutive weeks, or to be untreated. Primary end-points were cure, overall response rates and survival. Toxicity was also evaluated in these mice, and a USFDA general safety study was conducted in healthy Balb/c mice. In Raji cell culture, the threshold and IC50 concentrations for cytotoxic activity were 0.7 and 2.5 nmol (pm/ml media), respectively. When compared to treated Jurkat's xenografts or untreated xenografts, Raji xenografts treated with the SHAL showed an 85% reduction in hazard of death (P=0.014; 95% confidence interval 32-95% reduction). There was no evidence for toxicity even after i.p. doses 2000 times greater than the treatment dose associated with cure of a majority of the mice with Raji xenografts. When compared with control groups, treatment selectively improved response rates and survival in mice with HLA-DR10 expressing human lymphoma xenografts at doses not associated with adverse events and readily achievable in patients.

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules.

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study.

Semen samples from a fertile patient presenting with influenza and a 1-day fever of 39.9 degrees C were obtained and analyzed at 18-66 days postfever (dpf) for sperm nuclear proteins, DNA stainability, free thiols (-SH), and susceptibility to DNA denaturation in situ. At 18 dpf, 36% of sperm demonstrated denatured DNA as measured by the sperm chromatin structure assay (SCSA), and decreased to 23% by 39 dpf. Samples at 33 and 39 dpf contained 49% and 30%, respectively, of cells with increased DNA stainability (HIGRN). A unique sperm nuclear protein band migrating between histones and protamines on acid-urea gels appeared at 33 and 39 dpf and nearly disappeared by 52 dpf. Amino acid sequencing of the first 8 N-terminal residues identified this protein as the precursor to protamine 2. The protamine P1 and P2 ratio remained normal, whereas the histone to protamine ratio increased slightly at 33 to 39 dpf. Flow cytometric measurements of nuclear -SH groups revealed the greatest reduction in free nuclear thiols at 33 dpf, and returned to normal by 45 dpf. The time of appearance of the unprocessed protamine 2 precursor and the relative increase in histone suggest a fever-related disruption of the synthesis of mRNA that codes for a P2 processing enzyme or enzymes. Increased DNA staining is likely due to the increased histone/protamine ratio. This case study demonstrates that fever/influenza can have latent effects on sperm chromatin structure and may result in transient release of abnormal sperm.

Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2.

Although studies have demonstrated that zinc can bind to sperm nuclear proteins, specifically protamine 2, it has not been shown that the metal is sufficiently abundant inside the sperm nucleus to interact stoichiometrically with these proteins. In this study proton-induced X-ray emission (PIXE) has been used to measure the amount of sulfur and zinc within the nuclei of individual sperm cells to infer the stoichiometry of zinc binding to protamine 2 in six species of mammal: bull, chinchilla, stallion, hamster, human, and mouse (protamine 2 comprises from 0% (bull) to 67% (mouse) of the protamine present in the sperm of these animals). Using the sulfur mass and electrophoretic data on the relative proportion of protamine 1 and protamine 2 in the sperm chromatin of these species, the protamine 1, protamine 2, and total protamine contents within each species sperm nuclei have been determined. The PIXE measurements reveal that the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin. PIXE analyses of hamster protamines extracted under conditions that appear to at least partially preserve zinc binding also confirm that the majority of the metal is bound to protamine. In five of the species examined, sufficient zinc is present for each protamine 2 molecule to bind one zinc. The results obtained for chinchilla sperm, conversely, indicate the chinchilla protamine 2 molecule may interact differently with zinc. Chinchilla sperm only contain enough zinc for one atom to be bound to two protamine 2 molecules.

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A.

A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.

DNA condensation by protamine and arginine-rich peptides: analysis of toroid stability using single DNA molecules.

Both somatic cells and sperm have been shown to take up exogenous DNA, but the frequency of its integration is usually low. Scanning probe microscopy studies of sperm chromatin and synthetic DNA-protamine complexes indicate that the coiling of DNA into toroidal subunits, a process initiated in the maturing spermatid to prepare its genome for delivery into the egg, can be mimicked by simply adding protamine to DNA in vitro. The increased resistance of DNA-protamine complexes to nuclease digestion and their structural similarity to native sperm chromatin suggest that the packaging of DNA by protamine might offer a new approach for improving the efficiency of DNA uptake by sperm. Decondensation experiments performed with individual DNA molecules have provided a direct measure of the stability of toroids produced using salmon protamine and smaller arginine-rich peptides. These experiments show that the arginine content of protamine-related sequences can have a dramatic effect on their rate of dissociation from DNA. This technique and the information it provides can be used to identify protamine analogs that can be bound to DNA to increase the efficiency of its uptake by sperm and other cells.

Identification of novel small molecule ligands that bind to tetanus toxin.

Tetanus toxin belongs to a family of clostridial protein neurotoxins for which there are no known antidotes. Another closely related member of this family, botulinum toxin, is being used with increasing frequency by physicians to treat severe muscle disorders. Botulinum toxin has also been produced in large quantities by terrorists for use as a biological weapon. To identify small molecule ligands that might bind to the targeting domain of tetanus and botulinum toxins and to facilitate the design of inhibitors and new reagents for their detection, molecular docking calculations were used to screen a large database of compounds for their potential to bind to the C fragment of tetanus toxin. Eleven of the predicted ligands were assayed by electrospray ionization mass spectrometry (ESI-MS) for binding to the tetanus toxin C fragment, and five ligands (45%) were found to bind to the protein. One of these compounds, doxorubicin, was observed to have strong hydrophobic interactions with the C fragment. To check the ligands for their ability to compete with ganglioside binding, each was also tested using a GT1b liposome assay. Doxorubicin was the only ligand found to competitively bind the tetanus toxin C fragment with an appreciable binding constant (9.4 microM).

Cadmium concentrations in the testes, sperm, and spermatids of mice subjected to long-term cadmium chloride exposure.

BACKGROUND: Exposures to cadmium have been reported to reduce male fertility and there are several hypotheses that suggest how reduced male fertility may result from incorporation of cadmium into sperm chromatin. The purpose of this study was to determine whether mice subjected to long-term intraperitoneal cadmium exposure incorporated cadmium into their sperm chromatin. METHODS: Male mice were exposed to 0.1 mg/kg body weight cadmium in the form of CdCl2 via intraperitoneal injection once per week for 4, 10, 26, and 52 weeks and then sacrificed. The cadmium contents of the liver, testes, pooled sperm, and pooled spermatids from dosed and control animals were determined by atomic absorption spectroscopy. Cadmium and zinc contents in individual sperm and spermatid heads were determined by particle-induced x-ray emission. RESULTS: Atomic absorption spectroscopy revealed that although cadmium accumulated in the liver and testes, cadmium was not detected in pooled sperm or spermatid samples down to minimum detectable limits of 0.02 microg/g dry weight. Particle-induced x-ray emission analyses did not show the presence of cadmium in any sperm or spermatid head down to minimum detectable limits of 15 microg/g dry weight. Particle-induced x-ray emission analyses also demonstrated that phosphorus, sulfur, and zinc concentrations in individual sperm and spermatid heads were not altered by exposure to CdCl2. CONCLUSIONS: Because cadmium was not incorporated into sperm chromatin at levels above 0.02 microg/g dry weight, the data cast doubt on hypotheses that suggest that reduced male fertility may result from incorporation of cadmium into sperm chromatin.

Protamine-induced condensation and decondensation of the same DNA molecule.

The DNA in sperm and certain viruses is condensed by arginine-rich proteins into toroidal subunits, a form of packaging that inactivates their entire genome. Individual DNA molecules were manipulated with an optical trap to examine the kinetics of torus formation induced by the binding of protamine and a subset of its DNA binding domain, Arg6. Condensation and decondensation experiments with lambda-phage DNA show that toroid formation and stability are influenced by the number of arginine-rich anchoring domains in protamine. The results explain why protamines contain so much arginine and suggest that these proteins must be actively removed from sperm chromatin after fertilization.

Analysis of hamster protamines: primary sequence and species distribution.

Basic nuclear proteins were isolated from the sperm of the Syrian hamster Mesocricetus auratus and characterized by gel electrophoresis, amino acid analysis, and sequencing. Analyses of the proteins by gel electrophoresis show that sperm of this species contain both protamines 1 and 2. The two proteins were purified by HPLC and the complete primary sequence of hamster protamine 1 was determined by automated amino acid sequence analysis. The protein sequence was subsequently confirmed by sequencing the PCR-amplified protamine 1 gene. The first forty-two residues of the hamster protamine 2 sequence were obtained by amino acid sequence analysis of the isolated protein, and this sequence was also confirmed and extended by sequencing the gene. Total basic nuclear protein was also isolated from sperm of six other species of hamsters, the protamines were identified by HPLC and amino acid analysis, and the proportion of protamines 1 and 2 in each species was determined. Marked differences in the protamine 2 content of sperm were observed among the different species of hamster. This variation and the high level of sequence similarity between mouse and hamster protamines provide insight into how the two protamines may be organized in sperm chromatin. Mol. Reprod. Dev. 54:273-282, 1999. Published 1999 Wiley-Liss, Inc.

Protein and DNA contents in sperm from an infertile human male possessing protamine defects that vary over time.

Sperm from 2 semen samples collected 6 months apart from an infertile male and 3 semen samples collected over an 18-month period from a fertile human male volunteer have been analyzed for their protamine and DNA content. Hup1M and Hup2b antibodies were used to detect the presence of protamines and protamine precursors in western blots of nuclear proteins isolated from pools of sperm. Phosphorus and sulfur contents, which can be used to estimate the nuclear DNA and protamine contents of sperm from fertile males, were measured within individual sperm heads from each semen sample by particle induced x-ray emission (PIXE). The single-cell data reveal no significant differences in the phosphorus and sulfur contents of sperm heads in the three semen samples obtained from the fertile male. For the initial semen sample produced by the infertile male, Western blot data show a normal complement of protamine 1, small amounts of mature protamine 2, and reveal large amounts of anti-protamine 2 reactive proteins with electrophoretic mobilities similar to protamine 2 precursors. Data from PIXE show elevated levels of sulfur within sperm heads compared with sperm from the fertile male. Western blot data exhibit no evidence of protamines or protamine 2 precursors in the second semen sample produced by the infertile male. Data from PIXE suggest that these sperm are highly deficient in sulfur and protamines. These results show that the degree of maturation of sperm cells present in the semen of some infertile males can vary with time.

Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels.

OBJECTIVE: To determine whether the reduction in the protamine P2 content (increased P1/P2 ratio) reported in some infertile patients could result from incomplete processing of protamine P2 precursors. DESIGN: Analysis of samples with a marked reduction in the protamine P2 content using polyacrylamide gel electrophoresis and subsequent detection of protamine P2 precursors through Western blot analysis. SETTING: University departments and laboratories. PATIENT(S): One hundred eighty-four men undergoing an evaluation for infertility. MAIN OUTCOME MEASURE(S): Comparative Western blot analysis of nuclear sperm proteins using specific antibodies to protamine P1 and protamine P2. RESULT(S): After selection of the samples with a marked reduction of the protamine P2 content and subsequent analysis by Western blot, a small proportion of putative P2 precursors was detected in most samples, whereas a significant increase was detected in two of them. CONCLUSION(S): In some infertile men, a reduction in the protamine P2 content relative to protamine P1 (increased P1/P2 ratio) is detected concomitant with an increase in the amount of putative P2 precursors. This could represent the first report of incomplete processing of a nuclear sperm protein in humans.

Analysis of DNA-protamine interactions by optical detection of magnetic resonance.

Optically detected magnetic resonance (ODMR) has been used to identify the binding site of a synthetic protamine subdomain to the major groove of DNA. A 14 amino acid peptide (R6WGR6) analog of the central DNA binding domain of bull protamine was synthesized with phenylalanine replaced by tryptophan (Trp). The peptide was bound to double-stranded poly(dABrdU) and to calf thymus DNA (CT DNA) and the complexes characterized as "wet" solids using ODMR techniques. The appearance of the D + E transition in the slow passage ODMR and of short-lived components in the phosphorescence decay of the complex of R6WGR6 with poly(dABrdU) is diagnostic of a heavy atom effect. This can only occur if the peptide binds in the major groove of poly(dABrdU). The microenvironment of Trp in the nucleoprotein complex was characterized by phosphorescence, radiative decay lifetimes, and low-temperature ODMR measurements before and after binding to DNA. Bathochromic shifts in the phosphorescence emission upon exciting to the red in CT DNA-peptide suggest that the Trp is in a polar environment, while the red-shifted position of the 0, 0-band emission points to a more polarizable environment. The heavy atom effect strongly suggests a Trp location within the major groove of DNA. A partial stacking of Trp with the polarizable nucleobases and simultaneous interactions with the phosphate-guanidinium ion pairs and/or water molecules in the major groove of DNA which might not be totally displaced upon binding of the peptide could explain this conflicting evidence. Extrapolation of results from the system studied to protamine binding in sperm chromatin strongly suggests that the predominant binding site of protamine is the major groove of DNA.

AFM analysis of DNA-protamine complexes bound to mica.

A novel method for reconstituting sperm chromatin was used to investigate how protamine 1 condenses DNA. Complexes formed in vitro using linearized plasmid DNA were imaged and measured by atomic force microscopy (AFM). The structures formed were found to be highly dependent on the sample preparation method used for reconstitution. Interstrand, side-by-side fasiculation of DNA and toroidal-like structures only 1-2 DNA diameters thick were observed for complexes formed in solution following direct mixing of the DNA and protamine. Large chromatin aggregates were also observed on the mica. However, if the DNA was first allowed to attach to the mica prior to addition of the protamine, well-defined toroidal complexes were formed without any observed DNA fasiculation or aggregate formation. The diameter of the toroids measured 30.6-50.2 nm (mean 39.4 nm). The dimensions of these structures indicate that the condensed DNA is stacked vertically by four to five turns, with each coil containing as little as 360-370 bp of 'B'-form DNA. This approach for preparing and imaging DNA-protamine complexes permits the analysis of intermediate structures 'trapped' on the mica as partially formed toruses of nucleoprotamine.

Atomic force microscope analysis of chromatin volumes in human sperm with head-shape abnormalities.

We used the atomic force microscope to perform volume measurements on individual human sperm nuclei under ambient conditions. Data obtained for normal sperm and for sperm from seven of the nine classes of head-shape abnormality revealed that the nuclear volumes were essentially identical for sperm in each headshape class, even though the projected areas and shapes of the nuclei have were shown to vary widely. These results indicate that the abnormal sperm head morphologies found at a rate of 25-40% in fertile males are not caused by factors that affect the volume of sperm chromatin, such as the DNA content of the sperm nucleus, differences in chromatin organization, or the extent of DNA compaction.

Extent of sperm chromatin hydration determined by atomic force microscopy.

Volume measurements were performed on intact bull and mouse sperm heads and amembranous sperm nuclei, both in the fully hydrated (fluid cell) and dehydrated (air-dried on glass coverslips) states by atomic force microscopy (AFM). Data were obtained by analyzing a small population of cells/nuclei, as well as by performing repeated measurements on single cells imaged following the addition of increasing concentrations of propanol. Results show that the volume of fully hydrated, intact sperm heads and amembranous sperm chromatin particles are at least twice the volume of their air-dried counterparts. Dehydration occurs rapidly in air, and the reduction in volume of chromatin induced by water loss appears to be completely reversible. These studies demonstrate that both mouse and bull sperm chromatin are extensively hydrated in the native state, and are not as compact as previous studies have suggested.

Mapping and measuring DNA to protein ratios in mammalian sperm head by XANES imaging.

We have used XANES imaging, which combines X-ray absorption near edge spectral features (XANES) with 50-nm-resolution X-ray microscopy, to examine the content and distribution of DNA and protein in mature sperm cells. Sperm nuclei from five different species of mammals were examined; these species were chosen for analysis because their sperm contain marked differences in their protamine 1 and protamine 2 contents. The data we've obtained for bull, stallion, hamster, and mouse sperm suggest that the total nuclear protein to DNA ratio is similar in the sperm of many eutherian mammals. Since protamine constitutes the majority of the sperm nuclear protein, these results indicate that the total protamine content of sperm chromatin must be constant among mammalian species, independent of the extent of expression of the protamine 2 gene.

DNA and total protamine masses in individual sperm from fertile mammalian subjects.

The total amount of phosphorus and sulfur inside the nuclei of individual bull, stallion, hamster, human, and mouse sperm from fertile subjects has been measured using Particle Induced X-ray Emission (PIXE). Using the sulfur masses, we determined the total protamine (protamine 1 plus protamine 2) mass within the sperm nuclei of each species. Using the phosphorus masses, we determined the DNA mass present within the sperm nuclei of each species. The results reveal that although the relative proportion of protamine 1 to protamine 2 varies among the species examined, the total protamine mass to DNA mass ratio is similar in bull, stallion, hamster, and mouse sperm nuclei. In contrast, mature human sperm nuclei were found to contain significantly less protamine. This observation is consistent with other studies, which suggest that as much as 15% of the DNA in human sperm remain packaged by histones. Using the data obtained for bull sperm, the length of DNA that could be covered by each protamine 1 molecule in bull sperm has been estimated. Making the assumption that the size of the protamine 1 binding site on DNA is similar in the sperm of these species, the length of DNA covered by a single protamine 2 molecule also has been estimated.

The chromatin structure of well-spread demembranated human sperm nuclei revealed by atomic force microscopy.

The fundamental structure formed when genomic DNA is packaged by protamine in the human sperm nucleus still remains essentially unresolved. It is known that the binding of protamine, a small arginine-rich protein, to DNA generates a large dense, hydrophobic complex making the sperm chromatin structure difficult to study microscopically. To visualize the internal nuclear structures, isolated human sperm nuclei were swollen extensively in saline buffer using only a reducing agent. The nuclei were swollen during deposition onto coverglass and then imaged in the atomic force microscope (AFM). The two main results obtained from imaging individual well-spread nuclei indicate that native human sperm chromatin is: (1) particulate, consisting primarily of large nodular structures averaging 98 nm in diameter, and (2) also composed of smaller, nucleosome-like particles observed to form linear chains near the nuclear periphery. These two types of chromatin particles imaged by AFM are remarkably similar to other AFM measurements made on native and reconstituted sperm and somatic chromatin.

Well-defined genome architecture in the human sperm nucleus.

Using fluorescence in situ hybridization, conventional epifluorescence microscopy, and laser scanning confocal microscopy followed by three-dimensional reconstruction we describe a well-defined higher order packaging of the human genome in the sperm cell nucleus. This was determined by the spatial localization of centromere and telomere regions of all chromosomes and supported by localization of subtelomere sequences of chromosome 3 and the entire chromosome 2. The nuclear architecture in the human sperm is characterized by the clustering of the 23 centromeres into a compact chromocenter positioned well inside the nucleus. The ends of the chromosomes are exposed to the nuclear periphery where both the subtelomere and the telomere sequences of the chromosome arms are joined into dimers. Thus chromosomes in the human sperm nucleus are looped into a hairpin-like configuration. The biological implications of this nuclear architecture in spermatogenesis and male pronuclear formation following fertilization are discussed.