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The association of a pair of methane molecules in water has been investigated by molecular dynamics simulations with an optimized umbrella sampling method. The potential of mean force, entropy, and internal energy of the association have been calculated, in which for the first time the energetic contribution of water–water interaction to hydrophobic association was estimated. It turns out that the entropic contribution to the association is attractive at short distances and the energetic contribution of water–water interaction is very little. These results suggest that the number of hydrogen bondings between water molecules little change in the process of hydrophobic association.

In invertebrate species such as flies and nematodes, germline stem cells are maintained in a niche environment, which is restricted to the terminal end of the tubular structure in the gonads. In mice, spermatogonial stem cells (SSCs), a subpopulation of Asingle GFRα1 (glial cell line-derived neurotrophic factor [GDNF] family receptor-α1)-positive spermatogonia, are widely distributed along the longitudinal axis in the convoluted seminiferous tubules, preferentially juxtaposed to the interstitial vasculature. However, whether this area is the only SSC niche is not known. In this study, we identified a valve-like terminal segment of the seminiferous tubules, the Sertoli valve (SV), adjacent to the rete testis as another niche for GFRα1-positive spermatogonia in hamsters. Here, we show that the SV epithelium is composed of the modified Sertoli cells that are still capable of proliferation and missing most spermatogenic activities in the adult stage. The SV epithelium constitutively expresses GDNF, a major niche factor for SSCs, and supports the stable proliferation and selective maintenance of an Asingle subpopulation of GFRα1-positive spermatogonia in hamsters. The SV region of hamster seminiferous tubules has features that are similar to the stem cell niche in invertebrate gonads. Therefore, we propose that the SV may be a novel niche for Asingle GFRá1-positive spermatogonia potentially including a SSC population, at the terminal segments of the seminiferous tubules in hamsters. Stem Cells 2015;33:2811–2824

Electronic charged states of self-aligned coupled silicon quantum dots are investigated in this study using atomic force microscopy/Kelvin probe microscopy. Spatially-controlled charging characteristics of the quantum dot are studied. Results show that the temporal change in surface potential after electron injection is due to electron transfer in the quantum dot.

Many vasa homologue genes to Drosophila vasa have been isolated in various animal species. They provide specific molecular probes to analyze the establishment and the differentiation of germ cell lineage. In mammals, the expression of VASA protein becomes detectable in PGCs at the late migrating stage. Interestingly, during spermatogenesis the intracellular localization of VASA protein is closely associated with the chromatoid body.

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.

The morphological identification and ecological studies on the 3 subspecies of red jungle fowls were carried out in Thailand, Laos and Vietnam from 1994 to 1998. A total of 59 red jungle fowls and 47 hybrids with native chicken were collected and applied to the detailed morphological examination. For the ecological investigation, the hearing survey was performed at 55 localities. Fifty nine red jungle fowls were identified to 3 subspecies groups, Gallus gallus gallus with white earlobes, and G. g. spadiceus and G. g. jabouillei with red earlobes. The earlobe color was used as the most important index for the subspecies identification. Collecting places of individuals were also used as an effective index from the result of zoogeographical survey. There is an extensive zone of intergradation among the habitats of these 3 subspecies. The G. g. gallus inhabiting the continental Thailand, Cambodia, Laos and Vietnam expresses pure white earlobes covered completely with white patch, and the G. g. gallus inhabiting deep south Thailand expresses incomplete white earlobes.Therefore, the names of G. g. gallus type C (Continental) and G. g. gallus type I (insular) are reasonable to these zoogeographically differentiated two groups of G. g. gallus.

Hsc70t is a member of the Hsp70 family of genes and is constitutively expressed after meiosis in mouse spermatogenesis. Immunohistochemistry and in situ hybridization techniques were used to examine the precise localization of the Hsc70t product during the various stages of spermatogenesis. A rabbit antiserum raised againstthe mouse Hsc70t-lacZ fusion protein detected the Hsc70t protein in the late spermatid-enriched fraction after two-dimensional Western blot analyses. On histological sections, the protein appears in the cytoplasm of spermatids as they progress from step 9 to the final step of spermatogenesis. An antisense RNA probe generated from the 3' untranslated region of Hsc70t cDNA detected Hsc70t mRNA in late round spermatids from step 7 onward with the signal disappearing in spermatids at step 15. Thus, Hsc70t mRNA first appears after meiosis in haploid cells but is not translated effectively until these cells progress to the transcriptionally inactive stage which coincides with chromatin condensation. These results establish that the synthesis of Hsc70t protein is under strict translational control.

In the present study, genomic DNA was prepared from dried chicken blood sample and subsequently used for analyzing DNA fingerprinting (DFP) of native and Red jungle fowl in the South Pacific Islands. These chicken genomic DNAs were sufficient in quantity and purity for multiple DFP analyses, and chicken blood could be stored for up to 3 months without physical degradation under dry condition.The DFP analyses were carried out by individual and population DFP. Similarities of DFP patterns and genetic distances among fowls in Fiji and Western Samoa were estimated using band sharing (BS) value and the mean number of nucleotide substitutions per nucleotide site, respectively. The results of population DFP were under the influence of data in individual DFP. From the BS values and genetic distances, the native fowls in five different islands of Fiji and Western Samoa could be distinguished in each island, and the difference was consistent with geographical distribution. Fijian and Western Samoan native fowls were found to form two separate groups. The Red jungle fowls in Fiji islands were closely related to each other, but were found to be related remotely to Red jungle fowls in Kagoshima University. Therefore, DFP analysis can be used to estimate relative genetic variability and to reconstruct the evolutionary relationships in small populations genetically isolated.

In mammalian sex determination, SRY directly upregulates the expression of SOX9, the master regulatory transcription factor in Sertoli cell differentiation, leading to testis formation. Without SRY action, the bipotential gonadal cells become pre-granulosa cells, which results in ovarian follicle development. When, where and how pre-granulosa cells are determined to differentiate into developing ovaries, however, remains unclear. By monitoring SRY-dependent SOX9 inducibility (SDSI) in an Sry -inducible mouse system, we were able to identify spatiotemporal changes in the sexual bipotentiality/plasticity of ovarian somatic cells throughout life. The early pre-granulosa cells maintain the SDSI until 11.5 d.p.c., after which most pre-granulosa cells rapidly lose this ability by 12.0 d.p.c. Unexpectedly, we found a subpopulation of the pre-granulosa cells near the mesonephric tissue that continuously retains SDSI throughout fetal and early postnatal stages. After birth, these SDSI-positive pre-granulosa cells contribute to the initial round of folliculogenesis by the secondary follicle stage. In experimental sex reversal of 13.5-d.p.c. ovaries grafted into adult male nude mice, the differentiated granulosa cells re-acquire the SDSI before other signs of masculinization. Our data provide direct evidence of an unexpectedly high sexual heterogeneity of granulosa cells in developing mouse ovaries in a stage- and region-specific manner. Discovery of such sexually bipotential granulosa cells provides a novel entry point to the understanding of masculinization in various cases of XX disorders of sexual development in mammalian ovaries.

During mammalian sex determination of XY fetuses, SRY induces SOX9 in Sertoli cells, resulting in formation of testes with seminiferous tubules, interstitial Leydig cells and peritubular myoid cells. Meanwhile XX fetuses without SRY develop ovaries. In cattle, most XX heifers born with a male twin, so-called freemartins, develop nonfunctioning ovaries and genitalia with an intersex phenotype. Interestingly, freemartins sometimes develop highly masculinized gonads with seminiferous tubule-like structures despite the absence of SRY. However, in these cases, the degree of masculinization in each gonadal somatic cell type is unclear. Here, we report a rare case of a freemartin Japanese black calf with almost complete XX sexreversal. Gross anatomical analysis of this calf revealed the presence of a pair of small testis-like gonads with rudimentary epididymides, in addition to highly masculinized genitalia including a pampiniform plexus, scrotum and vesicular gland. Histological and immunohistochemical analyses of these masculinized gonads revealed well-defined seminiferous tubule-like structures throughout the whole gonadal parenchyma. In epithelia of these tubules, SOX9-positive supporting cells (i.e., Sertoli cells) were found to be arranged regularly along the bases of tubules, and they were also positive for GDNF, one of the major factors for spermatogenesis. 3β-HSD-positive cells (i.e., Leydig cells) and SMA-positive peritubular myoid cells were also identified around tubules. Therefore, for the first time, we found the transdifferentiation of ovarian somatic cells into all testicular somatic cell types in the XX freemartin gonads. These data strongly support the idea of a high sexual plasticity in the ovarian somatic cells of mammalian gonads.

Although di(n-butyl) phthalate (DBP), a suspected endocrine disruptor, induces testicular atrophy in prepubertal male rats, whether it exerts estrogenic activity in vivo remains a matter of debate. In the present study, we explored the estrogenic potency of DBP using 3-week-old male rats, and then examined the relationship between estrogen-induced spermatogenic cell apoptosis and testicular steroidogenesis. Daily exposure to DBP for 7 days caused testicular atrophy due to loss of spermatogenic cells, whereas testicular steroidogenesis was almost the same with the control values. A single exposure of DBP decreased testicular steroidogenesis in addition to decreasing the level of serum LH at 3 h after DBP treatment, with an extremely high incidence of apoptotic spermatogenic cells at 6 h after administration. To elucidate the estrogenic activity of DBP, we carried out an inhibition study using pure antiestrogen ICI 182,780 (ICI) in a model of spermatogenic cell apoptosis induced by DBP or estradial-3-benzoate (EB). Although both the DBP- and EB-treated groups showed a significant increase in spermatogenic cell apoptosis, ICI pretreatment significantly decreased the number of apoptotic spermatogenic cells in these two groups. In contrast, testicular steroidogenesis and serum FSH were significantly reduced in all the treated groups, even in the DBP+ICI and EB+ICI groups. Taken together, these findings led us to conclude that estrogenic compounds such as DBP and EB induce spermatogenic cell apoptosis in prepubertal rats, probably by activating estrogen receptors in testis, and that reduction in testicular steroidogenic function induced by estrogenic compounds is not associated with spermatogenic cell apoptosis.

Morphological alterations in seminiferous tubules caused by single administration of di(n-butyl) phthalate (DBP) in 3-week-old rats were investigated throughout the first wave of spermatogenesis. Single administration of DBP (500 mg/kg) showed progressive detachment and displacement of spermatogenic cells and disappearance of tubular lumen at 3 h after treatment, and then showed thin seminiferous epithelia and wide tubular lumen at day 1 (D1). At D1, quite significant numbers of apoptotic spermatogenic cells were detected, and then they gradually decreased in accordance with the passage of time. In contrast, the testes revealed lower weight gain, even after completion of first wave of spermatogenesis in the DBP-treated group, compared to the control. In order to clarify whether spermatogenic cells differentiate into mature spermatids in the DBP-treated rats, immunohistochemical staining for Hsc 70t, a specific marker for elongate spermatids, was carried out. As a result, the decrease in mature spermatids in the DBP-treated testes, compared to the control, was demonstrated. For example, at D20 (41-day-old) after treatment, the most advanced spermatids in the tubules from rats in the DBP-treated groups were steps 2–4, while those of the control were steps 12–13. Moreover, in some tubules, pachytene spermatocytes were the most advanced spermatogenic cell. At D30 (51-day-old) after treatment, maturation of spermatogenic cells in the DBP-treated rats proceeded further, and the most advanced spermatids in tubules were steps 8–9, while those of the control were steps 15–19. These results lead us to the postulation that a single administration of DBP to prepubertal rats delays maturation of spermatogenic cells, even after completion of first wave of spermatogenesis.

One of suspect environmental endocrine disruptors that affect mouse male reproduction by altering the morphology of Sertoli cells and spermatogenic cells is phthalate. The effects of mono(2-ethylhexyl)phthalate (MEHP), one of metabolites of di(2-ethylhexyl)phthalate , on immature mouse testes in vivo were examined. We have recently shown that MEHP induced Sertoli cells necrosis and spermatogenic cells apoptosis in mice by TUNEL method, F-actin staining, and ultrastructural study, but there is no data for biochemical changing of testes due to those methods could not explore. To verify in detail of it, we conducted Raman spectroscopy study with 785 nm wavelength laser line, 50mW of laser power and 3 minutes of exposure time to analysis the MEHP-treated testicular tissue, which has been fixatived by 4% paraformaldehyde (PFA). Five weeks old (5 w.o) male mice were used in this experiment. As the results, the alterations were observed by Raman spectroscopy that there are significantly differences of DNA, actin filament, type IV collagen and amide I between control group (0 μM MEHP) and treatment group (100 μM MEHP). These results significantly support histology staining observation (such as the apoptotic spermatogenic cells which is associated with DNA fragmentation and F-actin disruption) and ultrastructural observation (such as mitochondria rupture and disintegration of nucleus membrane). Raman spectroscopy can be used for 4% PFA-fixatived tissue observation. However, we recommend that Raman spectroscopy may be able to be expanded as an armamentarium not just for the clarification of histology staining and ultrastructural study, but furthermore, it may be as a non-invasion assessment for screening animal tissue toxicity of chemical in future.

Sox7, -17 and -18 constitute the Sox subgroup F (SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posterior dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.

Mono(2-ethylhexyl) phthalate (MEHP) is a well-characterized testicular toxicant. In this study, morphological alterations of mice testes caused by repeated administrations of MEHP were examined by light and transmission electron microscopy. Prepubertal male mice were given a range of MEHP doses (600-900 mg/kg/day) for 3 consecutive days in corn oil by oral gavage. Control animals were given only corn oil. Thereafter, the testes were excised, fixed in 4% paraformaldehyde for light microscopy and/or 5% glutaraldehyde for transmission electron microscopy. Then, they were embedded, and sectioned. TUNEL analysis was done to quantify the occurrence of apoptosis in the testis. Cellular damages were also observed. Results showed that administration of 700 mg/kg of MEHP caused a significant increase in TUNEL-positive cells. At the same time, mice treated with higher doses of MEHP showed presence of degenerating (apoptotic and necrotic) spermatogenic cells. Appearance of small vacuoles in the Sertoli cell cytoplasm and displacement of spermatogenic cells were also observed. Sloughed and shed spermatogenic cells found in the tubular lumen were identified to be necrotic and apoptotic in appearance, respectively.

The role of phagocytosis in eliminating apoptotic spermatogenic cells caused by mono(2-ethylhexyl) phthalate (MEHP) was studied. Twenty-one-day-old C57Bl/6N male mice were given a single dose of 800 mg/kg MEHP in corn oil by oral gavage and sacrificed at 1, 3, 5, 7 and 9 days after initial exposure. At the same time, the role of phagocytosis in MEHP related apoptosis was examined using microinjection of annexin V into the seminiferous tubules of living mice. Results showed that mice treated with MEHP had a lower rate of testis weight gain (lower regression line) and a significant TUNEL-positive spermatogenic cell number compared to control. However, this incident was reversible, and the number of TUNEL-positive cells returned to normal after 9 days. Mice microinjected with annexin V and later treated with MEHP showed a large amount of TUNEL-positive cells compared to mice treated with MEHP only. This clearly proves that phagocytosis plays an efficient and highly important role in eliminating dead cells in the injured testis of mice treated with MEHP.

The sex-determining region of Chr Y (Sry) gene is sufficient to induce testis formation and the subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males,such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth because of germ cell-autonomous defects. Therefore,it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here, we demonstrate that the testicular somatic environment of XX/Sry males is defective in supporting the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, of entering meiosis and of differentiating to the round-spermatid stage. XY-donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, resulting in severe deficiency of elongated spermatid stages. By contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY-donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed the missing expression of several Y-linked genes and the alterations in the expression profile of genes associated with spermiogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components,probably Sertoli cells, of XX/Sry testes, highlighting the idea that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice.

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7(-/-) mice. Transplantation of spermatogonial stem cells from male Cnot7(-/-) mice to seminiferous tubules of Kit mutant mice (Kit(W/W-v)) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7(-/-) condition. The testicular phenotypes of Cnot7(-/-) mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

To obtain a reliable molecular probe to trace the origin of germ cell lineages in birds, we isolated a chicken homolog (Cvh) to vasa gene (vas), which plays an essential role in germline formation in Drosophila. We demonstrate the germline-specific expression of CVH protein throughout all stages of development. Immunohistochemical analyses using specific antibody raised against CVH protein indicated that CVH protein was localized in cytoplasm of germ cells ranging from presumptive primordial germ cells (PGCs) in uterine-stage embryos to spermatids and oocytes in adult gonads. During the early cleavages, CVH protein was restrictively localized in the basal portion of the cleavage furrow. About 30 CVH-expressing cells were scattered in the central zone of the area pellucida at stage X, later 45-60 cells were found in the hypoblast layer and subsequently 200-250 positive cells were found anteriorly in the germinal crescent due to morphogenetic movement. Furthermore, in the oocytes, CVH protein was predominantly localized in granulofibrillar structures surrounding the mitochondrial cloud and spectrin protein- enriched structure, indicating that the CVH-containing cytoplasmic structure is the precursory germ plasm in the chicken. These results strongly suggest that the chicken germline is determined by maternally inherited factors in the germ plasm.

The anatomical location of testes in mammals ranges from a location close to that observed in the embryo to a lower position usually involving a pendant scrotum. In scrotal mammals, the abdominal position of the cryptorchid testis, which elevates its temperature, is detrimental to spermatogenesis and causes infertility. Spermatocytes are sensitive but late spermatids are relatively resistant to thermal stress suggesting that the latter might be protected in some way. In general, most organisms express Hsp70 proteins, which play a crucial role in the protection of cells against thermal stress. We have found previously that the Hsc70t protein, a member of the Hsp70 family of proteins, is constitutively expressed in the late spermatids of mice. Here, we have utilized immunohistochemistry with anti-mouse Hsc70t antiserum to examine the expression of the spermatid-specific Hsp70 antigen in the testes of several mammalian species with different degrees of testes migration. Our data indicate that the antigen is conserved in the mammals including marsupials. We also examined whether antigens of Hsp70-related proteins were expressed in non-mammalian vertebrates including not only homoiothermal but also poikilothermal animals. The spermatid-specific Hsp70 antigens were not detectable in the testes of the animals examined. From results of immunohistochemistry with BRM22 monoclonal antibody which reacts broadly with Hsp70 family proteins, however, we revealed constitutive expression of antigens of Hsp70-related proteins in spermatogenic cells of the vertebrates. These results suggest that the expression of spermatid-specific Hsp70 protein may be involved in the developmental pathway during spermiogenesis in mammals rather than in thermotolerance.

In mouse sex differentiation, SRY promotes Sertoli cell differentiation via SOX9 action, resulting in testis formation. SRY/SOX9 also initiates various testis-specific morphogenic events including glycogenesis in pre-Sertoli cells, suggesting the importance of glucose storage for certain SRY/SOX9-downstream events in gonadal sex determination. However, it remains unclear which cell types and what molecular/cellular events require sex-dimorphic high-energy metabolic rate. Here we show that the establishment of SOX9 activation itself is a metabolically active process with sex-dimorphic high-energy requirements in gonadal sex differentiation. The glucose-deprivation and metabolic rescue experiments using genital ridge cultures of the XY/XX-wildtype and XX/Sry transgenic embryos demonstrated that, among the various somatic cell types, pre-Sertoli cells are the most sensitive to glucose starvation despite the differences between XX/Sry and XY genotypes. Moreover, our data showed that, in developing pre-Sertoli cells, the high-glucose metabolic state is required for the establishment of SOX9 expression through an ECM (extracellular matrix)-mediated feed-forward pathway. In contrast, the expression of SRY, SF1/Ad4Bp, GATA4 and WT1, as well as initiation of early SOX9 expression, is properly maintained in the glucose-deprived condition. Therefore, our results imply the metabolic importance of the high-glucose condition for the establishment of SOX9 activation in testis differentiation.

The gonad as well as the reproductive tracts, kidney, and adrenal cortex are derived from the intermediate mesoderm. In addition, the intermediate mesoderm forms the mesonephros. Although the mesonephros is the source of certain testicular cell types, its contribution to gonad formation through expression of growth factors is largely unknown. Here, we examined the expression profiles of FGF9 in the developing mesonephros of chick embryos at sexually indifferent stages, and found that the expression domain is adjacent to the gonadal primordium. Moreover, FGFR3 (FGF receptor 3) showed a strong expression in the gonadal primordium. Next, we examined the functions of FGF signal during gonadal development with misexpressed FGF9. Interestingly, misexpression of FGF9 led to gonadal expansion through stimulation of cell proliferation. In contrast, treatment with a chemical inhibitor for FGFR decreased cell proliferation and resulted in reduction of the gonadal size. Simultaneously, the treatment resulted in reduction of gonadal marker gene expression. Our study demonstrated that FGF expressed in the developing mesonephros is involved in the development of the gonad at the sexually indifferent stages through stimulation of gonadal cell proliferation and gonadal marker gene expression.

Sodium-potassium ATPase transports three Na+ ions outward and two K+ ions inward by alternating two major conformational states, E1 and E2. There are three high affinity Na+ binding sites in E1 and two K+ binding sites in E2. These binding sites are located closely to each other in a transmembrane region containing several acidic residues. The protonation states of the acidic residues have been postulated to play an important role in ion selectivity. As the protonation states are strongly coupled, pKa estimations with programs, such as MCCE and PROPKA, using empirical point charges are ambiguous. It is questionable if conventional molecular dynamics simulations, though widely used, are applicable to determine the protonation states of strongly interacting residues, especially of protonated carboxyl groups. We therefore introduced quantum mechanical (QM) calculations in order to determine the positions of protons by calculating electrostatic potential (ESP) from electron densities. Starting with a model in which all acidic residues were deprotonated, the ESP showed several minima close to the carboxyl groups. The next model was generated by adding a proton at the minimum, and the ESP was calculated in the same way. Such procedure was repeated several times until no distinct minimum was found near the carboxyl groups. Finally, the geometry of the model was refined by QM to yield the model with explicit protons.