Nelson Hsia

THE TOXICITIES OF NATIVE AND MODIFIED HEMOGLOBINS

Recent research on the potential use of hemoglobin derivatives as a blood substitute has revealed that the administration of large quantities of free hemoglobin into the circulation results in a variety of toxic side effects. Because it has been well established that hemoglobin, like myoglobin, has considerable pro-oxidant activity, a number of studies have appeared suggesting that the administered hemoglobins may catalyze various oxidative and peroxidative reactions, which in turn, would cause the observed pathologic conditions. This occurs as a result of the in vivo formation of highly oxidized forms of the native and modified hemoglobins. In addition, it has been proposed that considerable amounts of free hemin and iron may be generated as a result of the catabolism of the injected hemoglobin. Hemin is known to be very toxic when present in large amounts, and iron could catalyze the formation of hydroxyl radicals via Fenton-type reactions. Thus, the toxic activities of catabolic products of hemoglobin could also be involved in all or part of the observed side effects. The purpose of this review is to consider the conditions under which reactive species of hemoglobin may be formed in vivo, their potential reactivity, and whether their individual or combined oxidative activities could account for the biological damage that is observed in vivo following hemoglobin transfusions.

A new subgroup of the family 2 cystatins.

The cystatins are a superfamily of cysteine protease inhibitors. Several genes including Cres (cystatin-related epididymal spermatogenic), testatin, and cystatin T, have been identified that are related to the family 2 cystatins but lack critical consensus sites important for cysteine protease inhibition. In addition, these genes are primarily expressed in the reproductive tract suggesting they may have evolved to perform tissue-specific functions distinct from that of the typical cystatins. This review describes the CRES subgroup within the family 2 cystatins including potential new members and their putative functions in the reproductive tract.

DNA Microarray Analysis of Region-Specific Gene Expression in the Mouse Epididymis

Abstract Microarray analysis was carried out to identify genes with enriched expression in the initial segment region of the mouse epididymis. A set of approximately 15 000 clones developed at the National Institutes for Aging and consisting of expressed sequence tags (ESTs) derived from pre- and peri-implantation embryos, Embryonic Day 12.5 female gonad/mesonephros, and newborn ovary were hybridized with probes generated against the initial segment (epididymal region 1) and the remainder of the epididymis (epididymal regions 2–5). The median values for the normalized ratios of region 1 to regions 2–5 from three independent experiments were averaged for each gene/EST using Genespring 5.0 software. The majority of clones showed a ratio of 1.0, suggesting they were expressed at similar levels in all epididymal regions. In addition, 123 clones exhibited 2-fold or higher expression in the initial segment, including Cres3, prostein, lipocalin 2, ALEX3, synaptotagmin-like 4, erm, and milk fat globule factor, whereas 216 clones, including elafin-like 1, lactotransferrin, Sin3B, zinc-finger protein 91, and membrane-type frizzled-related protein, showed 2-fold or higher expression in epididymal regions 2–5. Northern blot analyses of 12 clones predicted by microarray analysis to be either enriched in the initial segment (n = 8), enriched in epididymal regions 2–5 (n = 2), or similar in all regions (n = 2) were carried out. All clones exhibited the expected region-specific expression, thus confirming the microarray results. The studies presented here show a global survey of region-specific gene expression in the epididymis, identifying 15 287 sequences, the majority of which have not previously been shown to be expressed in this organ.

Gene and Protein Expression in the Epididymis of Infertile c-ros Receptor Tyrosine Kinase-Deficient Mice

Abstract Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein β, PEA3) were unchanged. Genes normally absent from the initial segment (γ-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.

Identification and Characterization of Cystatin-Related Epididymal Spermatogenic Protein in Human Spermatozoa: Localization in the Equatorial Segment

Abstract Our earlier studies in mouse have shown that the cystatin-related epididymal spermatogenic (CRES) protein is highly expressed in elongating spermatids in the testis and is present in mouse sperm acrosomes, suggesting specific roles in sperm function, fertilization, or both. However, whether the human CRES gene is similar to that of the mouse and is expressed in germ cells has not yet been determined. Therefore, the present study was undertaken to characterize the human ortholog of mouse Cres. Northern blot and in situ hybridization experiments showed that CRES is highly expressed in the human testis, specifically within clusters of round spermatids. Furthermore, reverse transcription-polymerase chain reaction detected CRES mRNA in the epididymis. Western blot analysis of protein lysates prepared from human testis and ejaculated spermatozoa showed a predominant 19-kDa protein and a minor 14-kDa protein. However, in contrast to the acrosomal localization of CRES protein in mouse spermatozoa, indirect immunofluorescence of human spermatozoa treated with methanol/acetic acid using anti-human CRES antibodies revealed that CRES was strictly localized to the equatorial segment. Furthermore, the same staining was observed in both capacitated and acrosome-reacted spermatozoa. To determine whether CRES was associated with the plasma membrane, live spermatozoa were incubated with CRES antibody after capacitation and acrosome reaction. Only acrosome-reacted spermatozoa showed a weak but specific equatorial staining. Taken together, these studies show that CRES protein is present in the sperm equatorial segment and becomes accessible to the extracellular environment during fertilization.

B-Myc, A Proximal Caput Epididymal Protein, Is Dependent on Androgens and Testicular Factors for Expression

Abstract The myc family of transcriptional regulators carries out critical roles in the control of cellular proliferation, differentiation, apoptosis, and tumorigenesis. The B-myc gene is a recently identified myc family member that has not been well characterized. Previously, we have shown that B-Myc inhibits the ability of c-Myc to transform cells and can inhibit cellular proliferation. Because B-myc is primarily expressed in hormonally regulated tissues with predominant expression in the epididymis, we examined in greater detail B-myc expression in the epididymis to ultimately understand potential roles B-myc may play in this and other hormonally regulated tissues. Herein we demonstrate that, in contrast to c-myc, B-myc mRNA and protein expression are highly regionalized with expression predominantly in the proximal caput epididymal region. Furthermore, in situ and immunohistochemical analyses show that within the epididymis B-myc mRNA and protein are specifically expressed by the epithelial cells and that B-Myc protein is localized to both the nuclear and cytosolic compartments. Castration and hormone replacement studies further show that expression of the B-myc mRNA is highly dependent on the presence of androgens and testicular factors. Finally, mRNA turnover studies demonstrate that the B-myc mRNA is relatively unstable with a half-life of 3.5 h. Taken together, the highly restricted and regulated expression of the B-myc gene suggests it may play important regulatory roles in the epididymis and perhaps other hormonally regulated tissues.

Characterization of Epididymal Epithelial Cell-Specific Gene Promoters by In Vivo Electroporation

Abstract The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and γ-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5′ site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between −530 and −681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.

CCAAT/Enhancer Binding Protein β Regulates Expression of the Cystatin-Related Epididymal Spermatogenic (Cres) Gene

Abstract The CRES protein is a member of the cystatin superfamily of cysteine protease inhibitors with restricted expression in stage-specific germ cells, proximal caput epididymidis, and anterior pituitary gonadotroph cells. To elucidate the molecular mechanisms regulating the highly restricted expression of the cres gene, we have sequenced 1.6 kilobases of mouse cres 5′ flanking sequence and performed studies to examine the cres gene promoter. Two putative CCAAT/enhancer binding protein (C/EBP) transcription factor binding motifs exist within the first 135 base pairs of cres promoter. Furthermore, our studies demonstrate that cres mRNA levels are dramatically reduced in the epididymides of C/EBPβ-deficient mice. These data suggest that the C/EBP family of transcription factors, in particular C/EBPβ, plays a role in the regulation of cres gene expression. In support of this finding, Northern blot analysis showed that C/EBPβ is the predominant C/EBP family member expressed in the LβT2 gonadotroph cell line and the proximal caput epididymidis. Also, gel shift and supershift assays demonstrated that C/EBPβ protein in nuclear extracts from LβT2 gonadotroph cells and epididymal cells bound to the two C/EBP sites in the cres promoter. Finally, to test the in vivo function of the C/EBP sites in cres gene expression, transfection studies were performed in LβT2 gonadotroph cells and two heterologous cell systems. These experiments showed a significant reduction of cres transactivation when either C/EBP sites were mutated, and no transC/EBP activation of the cres promoter when both C/EBP sites were mutated. Taken together, these studies demonstrate that the C/EBPβ transcription factor is necessary for high levels of cres gene expression in the proximal caput epididymidis and anterior pituitary gonadotroph cells.

The Cytotoxic Activities of Human Hemoglobin and Diaspirin Crosslinked Hemoglobin

It is well known that hemoglobin (Hb) possesses many oxidative enzyme activities, including a pseudo-peroxidase activity. It has also been shown by many investigators that various peroxidases in the presence of hydrogen peroxide and a halide ion exert a potent cytotoxic activity toward various mammalian cell types. It has further been observed by various investigators that the administration of relatively large amounts of purified Hb or a Hb derivative to a host animal during resuscitation experiments leads to a number of unrelated types of tissue damage and cell damage in the host. The first objective of this investigation was to determine if the observed tissue and cell damage may be due to a cytotoxic activity that Hb may exert in vivo analogous to that of the peroxidases. We also showed some time ago that peroxidases are able to activate peritoneal macrophages to the cytocidal state. Hence, we also addressed the question whether or not Hb is able to activate macrophages in a similar manner. Our results were negative with regard to both questions. Further investigations indicated that, unlike the peroxidases, ferryl-Hb is unable to oxidize iodide to iodine at a measurable rate, which appears to be the reason for the lack of cytotoxic activity.