Narrow Histopathological Margins are Acceptable in Surgical Resection of Basal Cell Carcinoma in Japanese: A Single-Center Retrospective Study.

BACKGROUND: Basal cell carcinoma (BCC) is the most common cutaneous malignancy. BCCs occur mainly in exposed areas, such as the face and scalp. Therefore, surgical resection with narrow margins is highly desirable. However, narrow margins may increase the risk of positive histopathological margins. Outcomes for such treatment might be unfavorable, but evidence for such a conclusion is lacking. METHODS: Between April 2015 and November 2023, a total of 230 Japanese cases with BCC which underwent surgical resection with 2-mm, 3-mm, or 5-mm margins were followed in our hospital. We conducted a retrospective review that focused on the recurrence rate and histopathological margins. RESULTS: Recurrence was recorded if the follow-up time was longer than 3 months. One of the 198 cases (0.5%) developed a recurrence. The mean lateral and deep histopathological margins were 2,525.4 µm (30.8-14,034.6 µm) and 3,409 µm (199.9-16,523.6 µm), respectively. Recurrence rate was associated with tumor size and clinical tumor border. However, histopathological margin was not associated with recurrence rate, even when it was less than 1,000 µm. CONCLUSIONS: A narrow histopathological margin is acceptable for surgical resection of BCC in Japanese patients.

Molecular basis for bacterial N-glycosylation by a soluble HMW1C-like N-glycosyltransferase.

Soluble HMW1C-like N-glycosyltransferases (NGTs) catalyze the glycosylation of Asn residues in proteins, a process fundamental for bacterial autoaggregation, adhesion and pathogenicity. However, our understanding of their molecular mechanisms is hindered by the lack of structures of enzymatic complexes. Here, we report structures of binary and ternary NGT complexes of Aggregatibacter aphrophilus NGT (AaNGT), revealing an essential dyad of basic/acidic residues located in the N-terminal all α-domain (AAD) that intimately recognizes the Thr residue within the conserved motif Asn0-X+1-Ser/Thr+2. Poor substrates and inhibitors such as UDP-galactose and UDP-glucose mimetics adopt non-productive conformations, decreasing or impeding catalysis. QM/MM simulations rationalize these results, showing that AaNGT follows a SN2 reaction mechanism in which the acceptor asparagine uses its imidic form for catalysis and the UDP-glucose phosphate group acts as a general base. These findings provide key insights into the mechanism of NGTs and will facilitate the design of structure-based inhibitors to treat diseases caused by non-typeable H. influenzae or other Gram-negative bacteria.

Change in Serum KL-6 Level during Biologic Treatment for Psoriasis.

BACKGROUND: We previously analyzed data from blood examination screenings, including serum Krebs von den Lungen (KL) -6 level, before starting biologic treatment for psoriasis in a real-world setting. However, we did not follow change in KL-6 level after the initiation of biologics. Furthermore, there has been no follow-up study of certolizumab pegol, risankizumab, or tildrakizumab. This study evaluated change in serum KL-6 levels in patients during treatment with biologics, including certolizumab pegol, risankizumab, and tildrakizumab. METHODS: We analyzed data from 111 patients. Change in KL-6 level was regarded as significant if it increased to greater than 500 U/mL at least once and if the maximum level after treatment with biologics was at least 1.5 times that of the baseline level. RESULTS: KL-6 level significantly changed during treatment with TNF inhibitors, IL-17 inhibitors, and IL-23 inhibitors in 9 (20.9%), 2 (6.3%), and 2 (5.6%) patients, respectively. Mean age, mean baseline KL-6 level, and frequency of TNF inhibitor use were higher in patients with a significant change in KL-6 level than those in patients without a significant change. Ten patients had minor interstitial changes on chest CT scans but no clinical signs suggesting interstitial pneumonia. CONCLUSIONS: Older patients with psoriasis and high baseline KL-6 levels must be carefully monitored during treatment with biologics, especially TNF inhibitors. Monitoring of KL-6 level and chest CT scans is necessary to exclude the possibility of drug-induced interstitial pneumonia.

Change in Antinuclear Antibody Titers during Biologic Treatment for Psoriasis.

BACKGROUND: We previously evaluated blood screening data, including antinuclear antibodies (ANA), before initiating biologic treatment for patients with psoriasis in a real-world setting. However, we did not analyze change in ANA titers after the start of biologics. No previous study has comprehensively investigated change in ANA titers over time in individual patients or the effectiveness of certolizumab pegol or tildrakizumab. OBJECTIVES: This study evaluated change in ANA titers in individual patients during treatment with biologics, including certolizumab pegol and tildrakizumab. METHODS: 111 patients were included in this study. Change in ANA was regarded as significant when the ANA titer was ×80 or more in patients with a previously undetectable ANA titer or when it increased by fourfold or more in those with a detectable ANA titer before treatment. RESULTS: The ratios of patients with a significant change in ANA titer who were treated with a tumor necrosis factor (TNF) inhibitor, interleukin (IL) -17 inhibitor, or IL-23 inhibitor were 34.9% (15/43), 0.0% (0/32), and 0.0% (0/36), respectively. There were 4 patterns of significant change in ANA titer: (i) an increase (n=8), (ii) a decrease after an increase (n=4), (iii) a decrease after an increase with a drug change (n=2), and (iv) an increase after a decrease after an increase (n=1). No symptom suggesting lupus syndrome was noted. CONCLUSIONS: ANA titers must be carefully monitored throughout treatment with biologics, especially TNF inhibitors, and the possibility of lupus-like syndrome should be excluded.

Bactericidal effect of lascufloxacin on HEp-2 cell-internalized group A Streptococcus.

INTRODUCTION: Although amoxicillin (AMPC) is recommended as first-line therapy for acute pharyngotonsillitis caused by group A streptococci (GAS), it often fails to eradicate infections. Internalization and subsequent intracellular survival of GAS are considered major mechanisms for penicillin therapeutic failure. It is, therefore, desirable to administer drugs that exert bactericidal effects on extracellular and intracellular GAS. In this study, we aim to investigate the bactericidal effects of lascufloxacin (LSFX) on internalized GAS in HEp-2 cells. MATERIALS AND METHODS: The GAS strain M1 and clinical isolate strain #2 were used in this study. Following treatment of GAS-infected human pharyngeal carcinoma epithelial HEp-2 cells with LSFX or AMPC, internalized GAS cells were recovered. The concentrations of LSFX and AMPC were equivalent to 1 × and 2 × MIC for strain M1. Culture medium was used as a control. Time-lapse and fluorescence images of GAS invading HEp-2 cell were obtained. LIVE/DEAD fluorescence staining was used to confirm the viability of internalized GAS. RESULTS: LSFX significantly reduced the number of cell-internalized M1 and #2 GAS strains compared to the control (p < 0.01) in a dose-dependent manner. However, AMPC did not reduce this in both strains. Both live and dead intracellular GAS were confirmed in HEp-2 cells exposed to LSFX. In contrast, intracellular GAS survived in HEp-2 cells exposed to AMPC and in the control. CONCLUSION: LSFX elicits significant bactericidal effects on cell-internalized GAS, hence it may represent a potent therapeutic option for patients with acute pharyngotonsillitis in whom AMPC treatment has failed.

Active Tuberculosis in a Patient Receiving Adalimumab for Psoriatic Arthritis and Chemoprophylaxis for Latent Tuberculosis Infection.

Tumor necrosis factor (TNF) inhibitors, including adalimumab, are widely used to treat refractory psoriatic arthritis (PsA). Although isoniazid chemoprophylaxis is generally effective in preventing reactivation of latent tuberculosis infection (LTBI), prophylactic measures do not fully protect against development of active tuberculosis. We report a rare case of active tuberculosis despite chemoprophylaxis for LTBI in a patient receiving adalimumab for PsA. A 60-year-old Japanese woman who had received a diagnosis of psoriasis at age 35 years presented with arthralgia of the right hand, which she first noticed 2 months previously. Physical examination showed scattered erythematous papules and plaques with scales on her trunk, extremities, and scalp. Her right metacarpophalangeal and proximal interphalangeal joints were swollen and painful, and her right wrist and elbow were painful. PsA was diagnosed and adalimumab was initiated. Because an interferon-γ release assay (IGRA) showed a borderline result at screening, isoniazid was administered as chemoprophylaxis for LTBI. At 22 months after initiation of adalimumab, IGRA was positive and chest CT disclosed centrilobular nodules in both lungs and swelling of multiple lymph nodes. Culture of sputum at 24 months demonstrated Mycobacterium tuberculosis. Active tuberculosis was diagnosed, and treatment with a combination of isoniazid, rifampicin, ethambutol hydrochloride, and pyrazinamide was started. To ensure timely diagnosis and treatment of active tuberculosis, a tuberculosis expert should be consulted at an early stage, with regular screening and monitoring.

Development of an amino acid sequence-dependent analytical method for peptides using near-infrared spectroscopy.

We aimed to develop an amino acid sequence-dependent analytical method using near-infrared (NIR) spectroscopy. The detailed analysis of the NIR spectra of eight different amino acid aqueous solutions (glycine, alanine, serine, glutamine, lysine, phenylalanine, tyrosine, and proline) revealed different spectral patterns characteristic of different amino acid residues in the 6200-5700 and 5000-4200 cm-1 regions, and the amino acids were identified based on the patterns. The spectra in the region of 5000-4500 cm-1 for tripeptide organic solutions that were composed of the aforementioned eight amino acids clearly showed the spectral differences depending on the amino acid species and amino acid sequences. Namely, tripeptide species were clearly differentiated from each other based on the spectral pattern of NIR bands due to the combinations of N-H stretching and amide II/III modes and those derived from the first overtones of amide II and amide I. The quantitative evaluation of changes in the concentrations of dipeptides and tripeptides composed of two different amino acids, glycine and proline was performed using partial least squares regression (PLSR) analysis and a combination of bands for amide modes. The calibration and validation results with high determination coefficients (R2 ≥ 0.99) were successfully obtained based on the amino acid sequences. The results not only revealed the usefulness of NIR spectroscopy as a process analytical technology (PAT) tool for synthesizing peptides in a micro flow reactor but also proposed a general method for quantitatively analyzing NIR spectra obtained in the course of chemical synthesis.

Two novel mutations in the ATP2C1 gene found in Japanese patients with Hailey-Hailey disease.

Hailey-Hailey disease (HHD) is an autosomal dominant genodermatosis and the defective gene in HHD is ATP2C1, which encodes secretory pathway Ca2+ /Mn2+ ATPase type 1 (SPCA1). Here we report four Japanese HHD patients showing three kinds of mutations with premature termination codons in the ATP2C1 gene, including two novel ones. Patient 1 was a 39-year-old man with a novel heterozygous mutation, c.664dup in exon 8 (p.N215Kfs*26). Patient 2 was a 33-year-old man (the younger brother of patient 1) with the same mutation as patient 1. Patient 3 was a 55-year-old man with a previously reported heterozygous mutation, c.519dup in exon 7 (p.R174Tfs*4). Patient 4 was a 33-year-old woman with a novel heterozygous mutation, c.2640del in exon 27 (p.L881Ffs*10). The clinical characteristics of our four cases varied in disease severity and the response to treatment. The present cases enrich the database of mutational analysis for HHD.

A case of pathologically confirmed streptococcal infection-related IgA vasculitis with associated glomerulonephritis and leukocytoclastic cutaneous vasculitis.

We report the case of an 80 year-old woman who developed bilateral lower extremity purpura and renal impairment with proteinuria a few days after a transient fever (day 0). High levels of both anti-streptolysin-O antibody (ASO) and anti-streptokinase antibody (ASK), as well as low levels of coagulation factor XIII in serum were noted. Skin biopsy was performed and showed a leukocytoclastic vasculitis with deposition of IgA and C3 in the cutaneous small vessels, indicating IgA vasculitis in the skin. After initiation of oral prednisolone, the skin lesions showed significant improvement. However, renal function and proteinuria gradually worsened from day 12. Kidney biopsy was performed on day 29, which demonstrated a necrotizing and crescentic glomerulonephritis with mesangial deposition of IgA and C3. In addition, the deposition of galactose-deficient IgA1 (Gd-IgA1) was positive on glomeruli and cutaneous small vessels, indicating that the purpura and glomerulonephritis both shared the same Gd-IgA1-related pathogenesis. In addition, the association between the acute streptococcal infection and the IgA vasculitis was confirmed by the deposition of nephritis-associated plasmin receptor (NAPlr) in glomeruli. The patient was treated with steroid pulse and intravenous cyclophosphamide, in addition to the oral prednisolone treatment. Renal function and proteinuria gradually improved, but did not completely recover, as is typically seen with courses of IgA vasculitis in the elderly. In this case, the streptococcal infectionrelated IgA vasculitis was confirmed pathologically by the deposition of both NAPlr and Gd-IgA1 in glomeruli, as well as Gd-IgA1 in the cutaneous small vessels.

Morphological and functional adaptation of pancreatic islet blood vessels to insulin resistance is impaired in diabetic db/db mice.

The pancreatic islet vasculature is of fundamental importance to the ß-cell response to obesity-associated insulin resistance. To explore islet vascular alterations in the pathogenesis of type 2 diabetes, we evaluated two insulin resistance models: ob/ob mice, which sustain large ß-cell mass and hyperinsulinemia, and db/db mice, which progress to diabetes due to secondary ß-cell compensation failure for insulin secretion. Time-dependent changes in islet vasculature and blood flow were investigated using tomato lectin staining and in vivo live imaging. Marked islet capillary dilation was observed in ob/ob mice, but this adaptive change was blunted in db/db mice. Islet blood flow volume was augmented in ob/ob mice, whereas it was reduced in db/db mice. The protein concentrations of total and phosphorylated endothelial nitric oxide synthase (eNOS) at Ser1177 were increased in ob/ob islets, while they were diminished in db/db mice, indicating decreased eNOS activity. This was accompanied by an increased retention of advanced glycation end-products in db/db blood vessels. Amelioration of diabetes by Elovl6 deficiency involved a restoration of capillary dilation, blood flow, and eNOS phosphorylation in db/db islets. Our findings suggest that the disability of islet capillary dilation due to endothelial dysfunction impairs local islet blood flow, which may play a role in the loss of ß-cell function and further exacerbate type 2 diabetes.

Method of Monitoring the Number of Amide Bonds in Peptides Using Near-Infrared Spectroscopy.

Using near-infrared (NIR) spectroscopy, we aimed to develop a method of monitoring the increasing number of amide bonds with the elongation of the chain length of peptides. Because peptide synthesis can be monitored by evaluating the increasing number of amide bonds with dehydration occurring between amino acids, polyglycine, which has the simplest structure among polyamino acids, was studied, and the key bands whose absorption intensities increased with the elongation of the chain length, such as the bands attributed to glycine, diglycine, triglycine, and tetraglycine, were searched. The bands due to the combinations of the amide A and amide II/III modes in the region of 5000-4500 cm-1 were revealed to be good candidates for key bands, their second derivative intensities increased as the number of amide bonds increased, regardless of pH, solvent species, and the presence of protecting groups. The number of amide bonds was evaluated by a partial least square regression using the abovementioned combination bands, and a calibration model with a high determination coefficient (≥0.99) was constructed. These results not only have demonstrated the usefulness of NIR spectroscopy as a process analytical technology tool for the process of synthesizing the peptide in a microflow reactor but also have provided basic knowledge for analyzing amide bonds in the NIR spectra of proteins, polyamino acids, polypeptides, and polyamides.

One-Step Synthesis of Sugar Nucleotides.

The chemical synthesis of sugar nucleotides requires a multistep procedure to ensure a selective reaction. Herein, sugar nucleotides were synthesized in one step using 2-chloro-1,3-dimethylimidazolinium chloride as the condensation reagent. The products were obtained in yields of 12-30%, and the yields were increased to 35-47% by the addition of a tuning reagent. NMR identification of the sugar nucleotides showed that mainly 1,2-trans-glycosides were present. The reported method represents a one-step route to sugar nucleotides from commercially available materials.

Long-distance proton transfer induced by a single ammonia molecule: ion mobility mass spectrometry of protonated benzocaine reacted with NH3.

Long-distance proton transfer is a ubiquitous phenomenon in chemical and biological systems. Two mechanisms of proton transfer in solids are well established; the Grotthuss mechanism (proton-relay) and the vehicle mechanism. Previously, intramolecular proton transfer has been extensively studied in the gas phase to understand the proton transfer mechanism microscopically. However, only the Grotthuss mechanism was proposed so far for intramolecular proton transfer. Here we show the first evidence for long-distance proton transfer (ca. 0.7 nm) via the vehicle mechanism in a gas-phase protonated molecule. Using ion mobility mass spectrometry, we observed that intramolecular proton transfer between two structural isomers with different protonation sites of protonated benzocaine (BC; p-NH2C6H4COOC2H5) is induced by a single NH3 molecule. In combination with theoretical calculations of the reaction pathway for the bimolecular reaction of BC·H+ + NH3, it was concluded that intramolecular proton transfer to produce the O-protomer (protonated BC at the C[double bond, length as m-dash]O group) proceeds in the N-protomer (protonated BC at the NH2 group) by NH3 coordination. In the calculated pathway, the NH4+ ion formed by proton transfer from the NH2 group of the N-protomer to NH3 donates a proton to the C[double bond, length as m-dash]O group after hopping on the benzene ring of BC. Our results demonstrate that we can investigate microscopically not only the Grotthuss mechanism but also the vehicle mechanism using gas-phase spectroscopic methods.

Polyfunctional Nanofibril Appendages Mediate Attachment, Filamentation, and Filament Adaptability in Leptothrix cholodnii.

Leptothrix is a species of Fe/Mn-oxidizing bacteria known to form long filaments composed of chains of cells that eventually produce a rigid tube surrounding the filament. Prior to the formation of this brittle microtube, Leptothrix cells secrete hair-like structures from the cell surface, called nanofibrils, which develop into a soft sheath that surrounds the filament. To clarify the role of nanofibrils in filament formation in L. cholodnii SP-6, we analyze the behavior of individual cells and multicellular filaments in high-aspect ratio microfluidic chambers using time-lapse and intermittent in situ fluorescent staining of nanofibrils, complemented with atmospheric scanning electron microscopy. We show that in SP-6 nanofibrils are important for attachment and their distribution on young filaments post-attachment is correlated to the directionality of filament elongation. Elongating filaments demonstrate a surprising ability to adapt to their physical environment by changing direction when they encounter obstacles: they bend or reverse direction depending on the angle of the collision. We show that the forces involved in the collision can be used to predict the behavior of filament. Finally, we show that as filaments grow in length, the older region becomes confined by the sheath, while the newly secreted nanofibrils at the leading edge of the filament form a loose, divergent, structure from which cells periodically escape.

Point Mutations Lead to Increased Levels of c-di-GMP and Phenotypic Changes to the Colony Biofilm Morphology in Alcanivorax borkumensis SK2.

Alcanivorax borkumensis is a ubiquitous marine bacterium that utilizes alkanes as a sole carbon source. We observed two phenotypes in the A. borkumensis SK2 type strain: rough (R) and smooth (S) types. The S type exhibited lower motility and higher polysaccharide production than the R type. Full genome sequencing revealed a mutation in the S type involved in cyclic-di-GMP production. The present results suggest that higher c-di-GMP levels in the S type control the biofilm forming behavior of this bacterium in a manner commensurate with other Gram-negative bacteria.

High glucose-mediated overexpression of ICAM-1 in human vaginal epithelial cells increases adhesion of Candida albicans.

To investigate the involvement of ICAM-1 in the adhesion of Candida to the genitourinary epithelial cells in high glucose, we examined the adhesion of Candida albicans or Candida glabrata to human vaginal epithelial cells (VK2/E6E7) or human vulvovaginal epidermal cells (A431). These cells were cultured in 100, 500 or 3000 mg/dL glucose for three days and inoculated with Candida for 60 minutes. Followed by, adhering of Candida to the cells, which were counted. While the adhesion of Candida albicans to VK2/E6E7 significantly increased in the high glucose, A431 did not. We next examined the expression of ICAM-1 as a ligand on the epithelial cells. ICAM-1 expression was increased in VK2/E6E7 cultured in the high glucose; however, the expression level in A431 was not high compared with VK2/E6E7. This data suggested that ICAM-1 functions as one of ligands in the adhesion of Candida albicans to the vaginal epithelial cells in a high glucose environment. Impact statement What is already known on the subject: Candida's complement receptor is involved in the adhesion to epithelial cells. The expression of this receptor has been reported to increase as glucose concentration increases. This is considered as a contributing factor to the high risk for vulvovaginal candidiasis (VVC) in diabetes. On the host side, diabetic patients have a factor that facilitates adhesion of Candida to epithelial cells. This factor has been unknown until recently. What the results of this study add: In this study, we used a vaginal epithelial cell line and showed that the adhesion of C. albicans to cells increased at higher glucose concentrations. At the same time, ICAM-1 expression of cells also increased. Thereby, it is suggested that the expression of ICAM-1 in vaginal epithelial cells is increased by glucose such as urinary sugar in diabetic patients and is a condition for facilitating adhesion of Candida. What the implications are of these findings for clinical practice and/or further research: We expect not only host immune dysfunction but also alteration in epithelial cells will be focussed on as a cause of VVC in diabetic patients.

Bazedoxifene Ameliorates Homocysteine-Induced Apoptosis and Accumulation of Advanced Glycation End Products by Reducing Oxidative Stress in MC3T3-E1 Cells.

Elevated plasma homocysteine (Hcy) level increases the risk of osteoporotic fracture by deteriorating bone quality. However, little is known about the effects of Hcy on osteoblast and collagen cross-links. This study aimed to investigate whether Hcy induces apoptosis of osteoblastic MC3T3-E1 cells as well as affects enzymatic and nonenzymatic collagen cross-links and to determine the effects of bazedoxifene, a selective estrogen receptor modulator, on the Hcy-induced apoptosis and deterioration of collagen cross-links in the cells. Hcy treatments (300 µM, 3 mM, and 10 mM) increased intracellular reactive oxygen species (ROS) production in a dose-dependent manner. Propidium iodide staining showed that 3 and 10 mM Hcy induced apoptosis of MC3T3-E1 cells. Moreover, the activities of caspases-8, 9, and 3 were increased by 3 mM Hcy. The detrimental effects of 3 mM Hcy on apoptosis and ROS production were partly reversed by bazedoxifene and 17ß estradiol. In addition, real-time PCR, immunostaining and Western blot showed that 300 µM Hcy decreased the expression of lysyl oxidase (Lox). Furthermore, 300 µM Hcy increased extracellular accumulation of pentosidine, an advanced glycation end product. Treatment with bazedoxifene ameliorated Hcy-induced suppression of Lox expression and increase in pentosidine accumulation. These findings suggest that high-dose Hcy induces apoptosis of osteoblasts by increasing oxidative stress, and low-dose Hcy decreases enzymatic collagen cross-links and increases pentosidine accumulation, resulting in the deterioration of bone quality. Bazedoxifene treatment effectively prevents the Hcy-induced detrimental reactions of osteoblasts. Thus, bazedoxifene may be a potent therapeutic drug for preventing Hcy-induced bone fragility.

Validation of the association between the gene encoding 5-lipoxygenase-activating protein and myocardial infarction in a Japanese population.

BACKGROUND: Recently, the 5-lipoxygenase activating protein gene (ALOX5AP) was reported to confer a risk of myocardial infarction (MI) and stroke, independent of conventional risk factors. The purpose of the present study was to validate those findings in a Japanese population. METHODS AND RESULTS: The study population consisted of 1,875 subjects (males 871, females 1,004) recruited from the Suita study (control group) and 353 subjects (males 306, females 47) with MI. The promoter, all of the exons, and 3'UTR regions of ALOX5AP were sequenced in 96 subjects, and 8 polymorphisms were found. There were significant differences in the frequencies of the haplotypes constructed from the 2 SNPs (A162C and T8733A) between the control and MI groups. Multiple logistic analysis indicated that the homozygous genotype of the (CA) haplotype was significantly associated with a reduced risk for MI. CONCLUSION: The hypothesis that ALOX5AP contributes to susceptibility for MI was validated in a Japanese population.

Calcium Wave in Activating Hamster Eggs.

The activating hamster egg shows a series of periodic increases in intracellular free calcium concentration ([Ca2+]i) (Igusa and Miyazaki, 1986). The spatial distribution of the Ca2+ transients can be analyzed in single, aequorin-injected eggs by the photon-counting imaging method with a supersensitive TV camera system (Miyazaki et al., 1986). A propagating increase in [Ca2+]i is observed in the first response, starting from the sperm attachment site and spreading over the entire egg within 4-7 s. The Ca wave is repeated in the second and sometimes the third response, starting from the same focus, but spreading more rapidly (∼2 s). In subsequent responses, [Ca2+]i increases synchronously in the whole egg within 1 s. A Ca2+ transient is induced in an all-or-none fashion by injection of either inositol 1,4,5-trisphosphate (IP3) or Ca2+ ions into unfertilized eggs. The Ca2+ transient is due to release of Ca2+ from intracellular stores, and it is followed by a refractory period of 60-120 s. Injection of guanosine-5'-0-(3-thiotriphosphate) (GTPγS) causes a series of Ca2+ transients with a pattern similar to that upon insemination, but with remarkable attenuation of the [Ca2+]i rise after 2-3 responses. Signal transduction involving activation of a GTP-binding protein and IP3- and/or Ca2+ -induced Ca2+ release is considered to be responsible for the repeated Ca2+ transients.