Comparison of dilute and nondilute osmotic equilibrium models for erythrocytes.

Successful cryopreservation requires the addition of cryoprotective agents (CPAs). The addition of permeating CPAs, such as glycerol, is associated with some risk to the cells and tissues. These risks are both related to the CPA themselves (CPA toxicity) and to the volume response of the cell (osmotic damage). To minimize the potential for damage during cryopreservation, mathematical models are often employed to understand the interactions between protocols and cell volume responses. In the literature, this volume response is usually captured using ideal and dilute approximations of chemical potential and osmolality, an approach that has been called into question for cells in high concentrations of CPAs. To address this, the relevance of non-ideal and non-dilute models has been explored in a number of cell types in the presence of permeating CPAs. However, it has not been explored in erythrocytes, which have a cytosolic hemoglobin content of more than 20% by volume and are cryopreserved in 40% glycerol. Because hemoglobin has been suggested to be a highly non-ideal solute, if the non-ideal and non-dilute transport model is relevant to any cells, it should be relevant to erythrocytes. Here we investigate the use, and accuracy, of both the dilute and non-dilute models in predicting cell volume changes during CPA equilibration in erythrocytes, and demonstrate that using published values for the non-ideal and non-dilute model, applied to erythrocytes, leads to model predictions inconsistent with experimental data, whereas dilute approximations align well with experimental data.

The cryobiology of spermatozoa.

The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm cryobiology, provide a synopsis of the development of sperm cryobiology as a discipline, and present the current state and directions for future research in sperm cryobiology in the three major areas outlined above-agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.

Papillomavirus E2 induces senescence in HPV-positive cells via pRB- and p21(CIP)-dependent pathways.

A hallmark of human papillomavirus (HPV) associated carcinogenesis is the integration of the viral DNA into the cellular genome, usually accompanied by the loss of expression of the viral E2 gene. E2 binds to and represses the viral promoter directing expression of the E6 and E7 oncogenes. The re-introduction and expression of exogenous E2 in HPV-positive cancer cells results in cellular growth arrest, while growth in the context of exogenous E2 can be restored through the expression of exogenous E6 and E7. Here we examine the individual contributions of the viral E6 and E7 genes to this phenotype. E6 alone displays moderate activity, whereas both E7 and adenovirus E1A display high activity in reversing E2-mediated cellular growth suppression. Using defined mutants of E7 and E1A, we show that an intact retinoblastoma interaction domain is required for this function. In addition, we show that the E2-mediated growth arrest of HPV-positive cells results in cellular senescence, and implicate the cyclin/cdk inhibitor p21(CIP) as a downstream E2 effector in this phenotype.

Effects of Percoll separation, cryoprotective agents, and temperature on plasma membrane permeability characteristics of murine spermatozoa and their relevance to cryopreservation.

Cryopreservation of murine spermatozoa would provide an efficient method for preserving important genotypes. However, to date such methods have resulted in low survivals with significant variability. To address this issue, a series of five experiments was performed to determine the cryobiological characteristics of murine spermatozoa. Experiments 1 and 2 investigated the effect of Percoll separation on the hydraulic conductivity (L(p)) of murine spermatozoa. Both Percoll separation and cryoprotective agents (CPAs) decreased the L(p). However, these effects were not additive. Experiment 3 was performed to determine the effect of temperature on L(p) in the presence of cryoprotectants (L(p)(CPA)), cryoprotectant permeability (P(CPA)), and the reflection coefficient (sigma) in spermatozoa from both ICR and B6C3F1 mice. Permeability parameters decreased as temperature decreased, and permeability characteristics differed between strains. In experiments 4 and 5, theoretical simulations for CPA addition and removal were developed and empirically tested. Strain-specific methods for CPA addition and removal based upon the fundamental cryobiological characteristics of murine spermatozoa resulted in higher survivals than current methods or procedures, which were used as controls.

Association of distinct yeast Not2 functional domains with components of Gcn5 histone acetylase and Ccr4 transcriptional regulatory complexes.

The NOT genes were originally identified in a yeast genetic screen that selected mutations resulting in increased utilization of a non-consensus TC TATA element of the HIS3 promoter. Here, we present evidence that the N-terminus of Not2 interacts with components of the Ada/Gcn5 histone acetyltransferase complex. Loss of this interaction either through abrogation of Not2 N-terminal function or deletion of ada2 or gcn5 results in derepression of the HIS3 TC element. This suggests that association of Not2 with the Ada/Gcn5 histone acetyltransferase complex is involved in regulation of the HIS3 promoter. Association between the Not and CCR4 transcriptional regulatory complexes has also been observed recently. Our phenotypic analyses suggest that these CCR4-related Not2 functions are mediated by a functionally independent domain of Not2 that includes the highly conserved C-terminus. Chimeric proteins containing the yeast Not2 N-terminus fused to the human C-terminus function in yeast, suggesting that the Not2 C-terminus represents a distinct modular domain whose function is conserved between higher and lower eukaryotes.

A fifteen-amino-acid peptide inhibits human papillomavirus E1-E2 interaction and human papillomavirus DNA replication in vitro.

Mutation of the conserved glutamic acid residue at position 39 of human papillomavirus type 16 (HPV-16) E2 to alanine (E39A) disrupts its E1 interaction activity and its replication function in transient replication assays but does not affect E2 transcriptional activation. This E39A mutation also disrupts replication activity of HPV-16 E2 in HPV-16 in vitro DNA replication. On this basis, we designed 23- and 15-amino-acid peptides derived from HPV-16 E2 sequences flanking the E39 residue and tested the ability of these peptides to inhibit interaction between HPV-16 E1 and E2 in vitro. The inhibitory activity of these peptides was specific, since analogous peptides in which alanine was substituted for the E39 residue did not inhibit interaction. The 15-amino-acid peptide E2N-WP15 was the smallest peptide tested that effectively inhibited HPV-16 E1-E2 interaction. This peptide also inhibited in vitro replication of HPV-16 DNA. The efficacy of E2N-WP15 was not exclusive to HPV-16: this peptide also inhibited interaction of HPV-11 E1 with the E2 proteins of both HPV-11 and HPV-16 and inhibited in vitro replication with these same combinations of E1 and E2 proteins. These results provide further evidence that E1-E2 interaction is required for papillomavirus DNA replication and constitute the first demonstration that inhibition of this interaction is sufficient to prevent HPV DNA replication in vitro.

Hydraulic conductivity (Lp) and its activation energy (Ea), cryoprotectant agent permeability (Ps) and its Ea, and reflection coefficients (sigma) for golden hamster individual pancreatic islet cell membranes.

Long-term cryopreservation of islets of Langerhans would be advantageous to a clinical islet transplantation program. Fundamental cryobiology utilizes knowledge of basic biophysical characteristics to increase the understanding of the preservation process and possibly increase survival rate. In this study several of these previously unreported characteristics have been determined for individual islet cells isolated from Golden hamster islets. Using an electronic particle counting device and a temperature control apparatus, dynamic volumetric response of individual islet cells to anisosmotic challenges of 1.5 M dimethyl sulfoxide (DMSO) and 1.5 M ethylene glycol (EG) were recorded at four temperatures (8, 22, 28, and 37 degreesC). The resulting curves were fitted using Kedem and Katchalsky equations which describe water flux and cryoprotectant agent (CPA) flux based on hydraulic conductivity (Lp), CPA permeability (Ps), and reflection coefficient (final sigma) for the membrane. For Golden hamster islet cells, Lp, Ps, and final sigma for DMSO at 22 degreesC were found to be 0.23 +/- 0.06 microm/min/atm, 0.79 +/- 0.32 x 10(-3) cm/min, and 0.55 +/- 0.37 (n = 11) (mean +/- SD), respectively. For EG at 22 degreesC, Lp equaled 0.23 +/- 0.06 microm/min/atm, Ps equaled 0.63 +/- 0.20 x 10(-3) cm/min, and final sigma was 0.75 +/- 0.17 (n = 9). Arrhenius plots (ln Lp or ln Ps versus 1/temperature (K)) were created by adding the data from the other three temperatures and the resulting linear regression yielded correlation coefficients (r) of 0.99 for all four plots (Lp and Ps for both CPAs). Activation energies (Ea) of Lp and Ps were calculated from the slopes of the regressions. The values for DMSO were found to be 12.43 and 18.34 kcal/mol for Lp and Ps (four temperatures, total n = 52), respectively. For EG, Ea of Lp was 11.69 kcal/mol and Ea of Ps was 20.35 kcal/mol (four temperatures, total n = 58).

Conserved interaction of the papillomavirus E2 transcriptional activator proteins with human and yeast TFIIB proteins.

Papillomavirus early gene expression is regulated by the virus gene-encoded E2 proteins. The best-characterized E2 protein, encoded by bovine papillomavirus type 1 (BPV-1), has been shown to interact with basal transcription factor IIB (TFIIB) and the TATA binding protein basal transcription factor (N. M. Rank and P. F. Lambert, J. Virol. 69:6323-6334, 1995). We demonstrate that the potent E2 transcriptional activator protein encoded by a gene of human PV type 16 also interacts with TFIIB in vitro. Moreover, a direct comparison of domains within human TFIIB (hTFIIB) required for VP16 and BPV-1 E2 indicates that these acidic activators interact with hTFIIB in a qualitatively similar manner. Our mapping experiments identify hTFIIB interaction domains within the amino-terminal activation domain of BPV-1 E2. Finally, we demonstrate in vitro interaction between Saccharomyces cerevisiae TFIIB and BPV-1 E2, an observation that is consistent with the importance of the E2-TFIIB interaction for BPV-1 E2 transactivation in both systems.

Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational constraints affecting E1 multimerization, E2 interaction, and interaction with cellular proteins.

Random mutagenesis of human papillomavirus type 16 (HPV16) E1 was used to generate E1 missense mutants defective for interaction with either hUBC9 or 16E1-BP, two cDNAs encoding proteins that have been identified by their ability to interact with HPV16 E1 in two-hybrid assays. hUBC9, the human counterpart of Saccharomyces cerevisiae UBC9, is a ubiquitin-conjugating enzyme known to be involved in cell cycle progression. 16E1-BP encodes a protein of no known function but does contain an ATPase signature motif. Eight hUBC9 or 16E1-BP interaction-defective HPV16 E1 missense mutants were identified and characterized for origin-dependent transient DNA replication, ATPase activity, and various protein-protein interaction phenotypes. Six of these mutant E1 proteins were significantly impaired for replication. Among these, two classes of replication-defective HPV16 E1 missense mutants were observed. One class, represented by the S330R replication-defective mutant (containing an S-to-R change at position 330), remained competent for all protein-protein interactions tested, with the exception of hUBC9 association. Furthermore, this mutant, unlike the other replication-defective HPV16 E1 missense mutants, had a strong dominant negative replication phenotype in transient-replication assays. The other class, represented by five of the missense mutants, was defective for multiple protein-protein interactions, usually including, but not limited to, the interaction defect for which each mutant was originally selected. In many cases, a single missense mutation in one region of HPV16 E1 had pleiotropic effects, even upon activities thought to be associated with other domains of HPV16 E1. This suggests that E1 proteins are not modular but may instead be composed of multiple structurally and/or functionally interdependent domains.

Mapping and characterization of the interaction domains of human papillomavirus type 16 E1 and E2 proteins.

The papillomavirus E1 and E2 proteins are both necessary and sufficient in vivo for efficient origin-dependent viral DNA replication. The ability of E1 and E2 to complex with each other appears to be essential for efficient viral DNA replication. In this study, we used the yeast two-hybrid system and in vitro binding assays to map the domains of the human papillomavirus type 16 (HPV16) E1 and E2 proteins required for complex formation. The amino-terminal 190-amino-acid domain of HPV16 E2 was both required and sufficient for E1 binding. The carboxyl-terminal 229 amino acids of E 1 were essential for binding E2, and the amino-terminal 143 amino acids of HPV16 E1 were dispensable. Although the ability of the E1 minimal domain (amino acids [aa] 421 to 649) to interact with E2 was strong at 4 degrees C, it was significantly reduced at temperatures above 25 degrees C. A larger domain of E1 from aa 144 to 649 bound E2 efficiently at any temperature, suggesting that aa 144 to 420 of E1 may play a role in the HPV16 E1-E2 interaction at physiological temperatures.

Cloning of the mouse fusin gene, homologue to a human HIV-1 co-factor.

Previous studies have demonstrated that mouse cells do not become infected with HIV-1 despite transfection with human CD4. Recently, a human protein termed "fusin" with characteristics of a seven-transmembrane-spanning receptor was found to be a co-factor required for the entry and fusion of HIV-1 with human CD4-bearing lymphocytes. Thus, cloning of the murine homologue of the human fusin (also termed CXCR-4) gene could provide an important comparative tool for identification of the structures crucial for fusin function. Using degenerate PCR, the mouse homologue of human fusin was cloned from a peritoneal exudate cell cDNA library. The predicted amino acid sequence is 91% identical to human fusin. Twenty-eight of the 37 amino acid differences between mouse and human fusin are located in the ectodomains, suggesting that the intracytoplasmic components that mediate G protein binding and signaling are highly conserved. Northern blot analysis showed a message of 2.2 kb in thymus, spleen, neutrophils, and primary astrocyte cultures. Lymphoid and monocyte cell lines also expressed message for fusin. The coding regions of most chemokine receptors lack introns. In contrast, cloning of genomic DNA for mouse fusin revealed the presence of a 2.3-Kb intron separating the first seven amino acids from the remaining 352 residues. Therefore, the mouse fusin gene has a unique genomic organization compared with other chemokine receptors.

Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions.

The E2 gene products of papillomavirus play key roles in viral replication, both as regulators of viral transcription and as auxiliary factors that act with E1 in viral DNA replication. We have carried out a detailed structure-function analysis of conserved amino acids within the N-terminal domain of the human papillomavirus type 16 (HPV16) E2 protein. These mutants were tested for their transcriptional activation activities as well as transient DNA replication and E1 binding activities. Analysis of the stably expressed mutants revealed that the transcriptional activation and replication activities of HPV16 E2 could be dissociated. The 173A mutant was defective for the transcriptional activation function but retained wild-type DNA replication activity, whereas the E39A mutant wild-type transcriptional activation function but was defective in transient DNA replication assays. The E39A mutant was also defective for HPV16 E1 binding in vitro, suggesting that the ability of E2 protein to form a complex with E1 appears to be essential for its function as an auxiliary replication factor.

Amino-terminal domains of the bovine papillomavirus type 1 E1 and E2 proteins participate in complex formation.

Interaction between the E1 and E2 papillomavirus proteins appear to play an important role in viral DNA replication, although the exact domains of each protein involved in this interaction have not been identified. Using bovine papillomavirus type 1 (BPV-1) as a model for examining interactions between E1 and E2, we have used the two-hybrid and glutathione S-transferase (GST) fusion systems to map domains of BPV-1 E1 and E2 that interact in vivo and in vitro. In the two-hybrid system experiments, portions of BPV-1 E2 were expressed in Saccharomyces cerevisiae as LexA fusion proteins, which were tested for interaction with various domains of BPV-1 E1. These assays indicated that domains sufficient for E1-E2 interaction are present within the amino-terminal 250 amino acids of E1 and within the first 91 amino acids of E2. Interestingly, a LexA fusion protein that included amino acid residues 53 to 161 of BPV E2 demonstrated transcriptional activation in this system. In vitro binding assays using combinations of BPV-1 E1-GST fusion proteins and BPV-1 E2 expressed by in vitro translation confirmed the observations from the yeast system; a GST fusion protein containing the first 222 amino acids of BPV-1 E1 bound specifically to full-length BPV-1 E2 in vitro. Furthermore, E1(1-222)-GST bound to forms of E2 deleted of the carboxy-terminal DNA binding-dimerization domain, suggesting that E2 dimerization is not required for this interaction. Finally, in vitro interaction between E1-GST and E2 was observed at 22 degrees C but not at 4 degrees C.

Abuse and HIV-related risk.

AIDS: Research has been conducted on the effects of childhood abuse that may lead to HIV risk-taking behaviors in adolescence and adulthood. There is evidence of a higher incidence of childhood abuse among HIV-infected individuals and those at highest risk, but not enough evidence to confirm childhood abuse as a root cause of risk-taking. Research is beginning to show important connections between psychological trauma and risk, and to suggest therapeutic approaches. The trauma of childhood sexual abuse is retained by the victims, which can cause dissociative defenses and damaging feelings of self-efficacy which may affect safer sex practices. To identify sexual abuse as an issue in therapy, it is advised that counselors develop client histories regarding relationships, sexual expression and identity, substance use, suicidality, and other recurring self-destructive patterns of thought and behavior. The treatment goal is to assist the patient in understanding how abuse has affected feelings, thoughts, and relationship styles, and to recognize patterns that lead directly to HIV-related risk or that set the stage for risky activities. The critical aspect of treatment is to accurately assess the patient's coping skills when determining the course of treatment. ¿¿¿¿¿¿¿^ieng

Infant formula development: past, present and future.

Currently available infant formulas can be separated into those intended for normal term infants and those designed for infants with special needs, i.e. infants with low-birth-weight, with allergies to milk proteins, or with metabolic disorders. New formulas are developed when groups of infants with special nutritional needs are identified. A recent example is the introduction of a soy fiber-containing formula for refeeding infants after diarrhea. Existing formulas continually change with new nutritional knowledge; an example is the addition of taurine when its role is visual function became known. The composition of human milk serves as a valuable reference for improving infant formula. However, human milk contains living cells, hormones, active enzymes, immunoglobulins and components with unique molecular structures that can not be replicated in infant formula. Additionally, unlike human milk, infant formula must remain stable on the shelf for up to 36 months. These fundamental differences between human milk and infant formula often mandate differences in composition to achieve similar clinical outcomes. New formulas or changes in formulas should confer a demonstrable advantage to the infant and not be based on compositional changes alone. Before changes are made in formulations or new formulas developed, a thorough assessment of available research needs to be made and any gaps of knowledge identified. Then a research program specific for the question at hand is developed.(ABSTRACT TRUNCATED AT 250 WORDS)

Docosahexaenoic acid status of term infants fed breast milk or infant formula containing soy oil or corn oil.

The objective of this study was to compare circulating lipid docosahexaenoic acid [22:6(n-3), DHA] levels in term infants fed a powdered (CORN oil) or liquid (SOY oil) infant formula or human milk (HM). Infants whose mothers chose not to breast feed were randomly assigned to the CORN or SOY formula group. The formula fat differed in linolenic acid [18:3(n-3)] content: it was 0.8% for the CORN and 4.8% for the SOY. Linoleic acid [18:2(n-6)] was 31.5 and 34.2% fatty acids in the CORN and SOY formula, respectively. The formulas or HM were fed from birth through 8 wk of age, and growth and the plasma and red blood cell (RBC) phospholipid fatty acid composition was determined at 3 d, 4 wk, and 8 wk of age. Growth did not differ among groups. The plasma phospholipid and RBC phosphatidylethanolamine DHA was similar in the CORN and SOY formula groups at all ages. Plasma and RBC phosphatidylethanolamine levels of DHA were significantly lower in infants fed the CORN or SOY formula than in infants fed HM during wk 4 and 8. Plasma and RBC 22:5(n-6) was not increased in the formula groups at any age. The formula content of linolenic acid had no effect on the RBC or plasma DHA levels of the infants. The biologic or functional significance of the lower plasma and RBC DHA in infants fed formula rather than HM is unknown. The need for a dietary source of DHA and specificity of plasma or RBC phospholipid DHA as a measure of desaturation and elongation of linolenic acid in developing organs remains uncertain.

Insulin-like growth factors I and II expression in the healing wound.

Demonstrating temporal variation in the expression of messenger RNA (mRNA) for growth factors may give some indication as to whether growth factor synthesis is regulated in wound healing. The aim of this study was to evaluate the expression of insulin-like growth factors (IGF) I and II in the wound. Two wound models, an incisional model and a subcutaneous sponge implant model, were used in this study. The RNA was extracted and reverse transcribed and mRNA was amplified using polymerase chain reaction (PCR). Semiquantitation of PCR products was accomplished using [3H]dGTP incorporation. Levels of expression for both IGF-I and -II were found to be low in unwounded skin and at 12 hr postwounding. However, in both wound models expression increased substantially from 1 to 21 days postwounding. Both factors also were found to be expressed by fibroblasts and polymorphonuclear leukocytes (PMN). Additionally, two transcripts were found for IGF-II, the larger of which appeared to be specific for PMN and possibly cells involved in angiogenesis. Levels of message expressed in healing wounds for IGF-I and -II appear to be regulated with the highest levels of message found at time points coinciding with fibroblast predominance in the wound. Since fibroblasts are known to both secrete and respond to IGF-I, it is possible that IGF-I and IGF-II are acting to influence fibroblast differentiation and function in the later stages of wound healing.

Irreversible inhibition of human cytomegalovirus replication by topoisomerase II inhibitor, etoposide: a new strategy for the treatment of human cytomegalovirus infection.

We demonstrated previously that human cytomegalovirus (CMV) infections could enhance the expression of cellular topoisomerase II and this enzyme activity is essential for CMV to replicate in vitro (Benson and Huang, 1988; Benson and Huang, 1990). In this study, we further show that in addition to m-AMSA and VM26 which we had previously reported, a widely used and clinically available drug, etoposide (VP-16 or VePesid) can irreversibly inhibit CMV replication at the drug concentration (2.5 micrograms/ml) greatly below toxic levels to stationary phase cells. Growing cells were more sensitive to etoposide than stationary phase cells and slight growth inhibition occurred at 2.5 micrograms/ml level. This inhibitor does not prevent the expression of CMV immediate-early and early genes, but can inhibit viral DNA and late viral-proteins synthesis. Because of their irreversible inhibitory effects and approval usage in clinical oncology, it is suggested that this group of compounds, particularly etoposide (VP-16), can be used to control life-threatening CMV infections, such as CMV pneumonitis and CMV retinitis, in cancer and immunocompromised patients or patients with AIDS.

Phaeohyphomycotic brain abscess due to Ochroconis gallopavum in a patient with malignant lymphoma of a large cell type.

A 60-year-old man with a 9-year history of malignant lymphoma developed an initial pulmonary infection with Nocardia asteroides which later disseminated to the central nervous system with multiple brain abscesses. He was treated successfully with intravenous trimethoprim-sulfamethoxazole for 6 weeks. A follow-up computed tomography (CT) scan showed complete resolution of the abscesses. Two years later, he returned to the hospital with a 2-week history of confusion, loss of concentration, ataxia, and leaning to the left. A CT scan revealed an enhancing multiloculated complex right frontal lesion. Craniotomy revealed a large right frontal lobe abscess, which was totally resected. Histopathologic examination of the resected tissue revealed multiple, lightly pigmented, septate, branched hyphal elements typical of phaeohyphomycosis. The fungal isolate cultured from the tissue was a dematiaceous, thermotolerant fungus that was identified as Ochroconis gallopavum. Despite treatment with amphotericin B, flucytosine and fluconazole, the patient gradually deteriorated and died. This case represents the third fatal infection, the second from the southeastern United States, due to O. gallopavum.