Photo-induced processes in photosynthesis-from femtoseconds to seconds.

Photosynthesis nourishes nearly all life on Earth. Therefore, a deeper understanding of the processes by which sunlight is converted into stored chemical energy presents an important and continuing challenge for fundamental scientific research. This Special Issue is dedicated to academician Vladimir A. Shuvalov (1943-2022). We are delighted to present 15 manuscripts in the Special Issue, including two review articles and 13 research papers. These papers are contributed by 67 authors from 8 countries, including China (9), Germany (8), Hungary (4), Italy (6), Japan (2), Russia (24), Taiwan (9), and USA (5). This Special Issue provides some of the recent updates on the dynamical aspects of the initial steps of photosynthesis, including excitation energy transfer, electron transport, and dissipation of energy across time domains from femtoseconds to seconds. We hope that the readers will benefit from the work presented in this Special Issue in honor of Prof. Shuvalov in many ways. We hope that the Special Issue will provide a valued resource to stimulate research efforts, initiate potential collaboration, and promote new directions in the photosynthesis community.

Revelations on photosystem II, thermoluminescence, and artificial photosynthesis: a retrospective of Govindjee from fundamentals to applications.

Photosynthesis, as one of the most important chemical reactions, has powered our planet for over four billion years on a massive scale. This review summarizes and highlights the major contributions of Govindjee from fundamentals to applications in photosynthesis. His research included primary photochemistry measurements, in the picosecond time scale, in both Photosystem I and II and electron transport leading to NADP reduction, using two light reactions. He was the first to suggest the existence of P680, the reaction center of PSII, and to prove that it was not an artefact of Chlorophyll a fluorescence. For most photobiologists, Govindjee is best known for successfully exploiting Chlorophyll a fluorescence to understand the various steps in photosynthesis as well as to predict plant productivity. His contribution in resolving the controversy on minimum number of quanta in favor of 8-12 vs 3-4, needed for the evolution of one molecule of oxygen, is a milestone in the area of photosynthesis research. Furthermore, together with Don DeVault, he is the first to provide the correct theory of thermoluminescence in photosynthetic systems. His research productivity is very high: ~ 600 published articles and total citations above 27,000 with an h-index of 82. He is a recipient of numerous awards and honors including a 2022: Lifetime Achievement Award of the International Society of Photosynthesis Research. We hope that the retrospective of Govindjee described in this work will inspire and stimulate the readers to continue probing the photosynthetic apparatuses with new discoveries and breakthroughs.

Polyphosphate kinase deletion increases laboratory productivity in cyanobacteria.

Identification and manipulation of cellular energy regulation mechanisms may be a strategy to increase productivity in photosynthetic organisms. This work tests the hypothesis that polyphosphate synthesis and degradation play a role in energy management by storing or dissipating energy in the form of ATP. A polyphosphate kinase (ppk) knock-out strain unable to synthesize polyphosphate was generated in the cyanobacterium Synechocystis sp. PCC 6803. This mutant strain demonstrated higher ATP levels and faster growth than the wildtype strain in high-carbon conditions and had a growth defect under multiple stress conditions. In a strain that combined ppk deletion with heterologous expression of ethylene-forming enzyme, higher ethylene productivity was observed than in the wildtype background. These results support the role of polyphosphate synthesis and degradation as an energy regulation mechanism and suggest that such mechanisms may be effective targets in biocontainment design.

Bimodal intramolecular excitation energy transfer in a multichromophore photosynthetic model system: hybrid fusion proteins comprising natural phycobilin- and artificial chlorophyll-binding domains.

The phycobilisomes of cyanobacteria and red-algae are highly efficient peripheral light-harvesting complexes that capture and transfer light energy in a cascade of excitation energy transfer steps through multiple phycobilin chromophores to the chlorophylls of core photosystems. In this work, we focus on the last step of this process by constructing simple functional analogs of natural phycobilisome-photosystem complexes that are based on bichromophoric protein complexes comprising a phycobilin- and a chlorophyll- or porphyrin-binding domain. The former is based on ApcE(1-240), the N-terminal chromophore-binding domain of the phycobilisome's L(CM) core-membrane linker, and the latter on HP7, a de novo designed four-helix bundle protein that was originally planned as a high-affinity heme-binding protein, analogous to b-type cytochromes. We fused a modified HP7 protein sequence to ApcEΔ, a water-soluble fragment of ApcE(1-240) obtained by excising a putative hydrophobic loop sequence of residues 77-153. HP7 was fused either to the N- or the C-terminus of ApcEΔ or inserted between residues 76 and 78, thereby replacing the native hydrophobic loop domain. We describe the assembly, spectral characteristics, and intramolecular excitation energy transfer of two unique systems: in the first, the short-wavelength absorbing zinc-mesoporphyrin is bound to the HP7 domain and serves as an excitation-energy donor to the long-wavelength absorbing phycocyanobilin bound to the ApcE domain; in the second, the short-wavelength absorbing phycoerythrobilin is bound to the ApcE domain and serves as an excitation energy donor to the long-wavelength absorbing zinc-bacteriochlorophyllide bound to the HP7 domain. All the systems that were constructed and tested exhibited significant intramolecular fluorescence resonance energy transfer with yields ranging from 21% to 50%. This confirms that our modular, covalent approach for studying EET between the cyclic and open chain tetrapyrroles is reasonable, and may be extended to larger structures mimicking light-harvesting in cyanobacteria. The design, construction, and characterization process demonstrated many of the advances in constructing such model systems, particularly in our ability to control the fold and aggregation state of protein-based systems. At the same time, it underlines the potential of exploiting the versatility and flexibility of protein-based systems in assembling multiple pigments into effective light-harvesting arrays and tuning the spectral properties of multichromophore systems.

Response of chlorophyll d-containing cyanobacterium Acaryochloris marina to UV and visible irradiations.

We have previously investigated the response mechanisms of photosystem II complexes from spinach to strong UV and visible irradiations (Wei et al J Photochem Photobiol B 104:118-125, 2011). In this work, we extend our study to the effects of strong light on the unusual cyanobacterium Acaryochloris marina, which is able to use chlorophyll d (Chl d) to harvest solar energy at a longer wavelength (740 nm). We found that ultraviolet (UV) or high level of visible and near-far red light is harmful to A. marina. Treatment with strong white light (1,200 µmol quanta m(-2) s(-1)) caused a parallel decrease in PSII oxygen evolution of intact cells and in extracted pigments Chl d, zeaxanthin, and α-carotene analyzed by high-performance liquid chromatography, with severe loss after 6 h. When cells were irradiated with 700 nm of light (100 µmol quanta m(-2) s(-1)) there was also bleaching of Chl d and loss of photosynthetic activity. Interestingly, UVB radiation (138 µmol quanta m(-2) s(-1)) caused a loss of photosynthetic activity without reduction in Chl d. Excess absorption of light by Chl d (visible or 700 nm) causes a reduction in photosynthesis and loss of pigments in light harvesting and photoprotection, likely by photoinhibition and inactivation of photosystem II, while inhibition of photosynthesis by UVB radiation may occur by release of Mn ion(s) in Mn4CaO5 center in photosystem II.

Methodology of pulsed photoacoustics and its application to probe photosystems and receptors.

We review recent advances in the methodology of pulsed time-resolved photoacoustics and its application to studies of photosynthetic reaction centers and membrane receptors such as the G protein-coupled receptor rhodopsin. The experimental parameters accessible to photoacoustics include molecular volume change and photoreaction enthalpy change. Light-driven volume change secondary to protein conformational changes or electrostriction is directly related to the photoreaction and thus can be a useful measurement of activity and function. The enthalpy changes of the photochemical reactions observed can be measured directly by photoacoustics. With the measurement of enthalpy change, the reaction entropy can also be calculated when free energy is known. Dissecting the free energy of a photoreaction into enthalpic and entropic components may provide critical information about photoactivation mechanisms of photosystems and photoreceptors. The potential limitations and future applications of time-resolved photoacoustics are also discussed.

Structural and mechanistic aspects of Mn-oxo and co-based compounds in water oxidation catalysis and potential applications in solar fuel production.

To address the issues of energy crisis and global warming, novel renewable carbon-free or carbon-neutral energy sources must be identified and developed. A deeper understanding of photosynthesis is the key to provide a solid foundation to facilitate this transformation. To mimic the water oxidation of photosystem II oxygen evolving complex, Mn-oxo complexes and Co-phosphate catalytic material were discovered in solar energy storage. Building on these discoveries, recent advances in solar energy conversion showed a compelling working principle by combing the active Mn-oxo and Co-based catalysts in water splitting with semiconductor hetero-nanostructures for effective solar energy harnessing. In this review the appealing systems including Mn-oxo tetramer/Nafion, Mn-oxo dimer/TiO(2), Mn-oxo oligomer/WO(3), Co-Pi/Fe(2)O(3), and Co-Pi/ZnO are summarized and discussed. These accomplishments offer a promising framework and have a profound impact in the field of solar fuel production.

Thermodynamics of charge separation of photosystem I in the menA and menB null mutants of Synechocystis sp. PCC 6803 determined by pulsed photoacoustics.

When the biosynthesis of phylloquinone is inhibited in Synechocystis sp. PCC 6803 by interrupting the menA or the menB gene, photosystem I (PS I) recruits plastoquinone-9 (A(P)) to occupy the A(1) sites. In PS I from the menA and menB null mutants, forward electron transfer from the quinone to the FeS clusters occurs approximately 1000 times slower than in wild-type PS I [Semenov, A. Yu., Vassiliev, I. R., van der Est, A., Mamedov, M. D., Zybailov, B., Shen, G., Stehlik, D., Diner, B. A., Chitnis, P. R., and Golbeck, J. H. (2000) J. Biol. Chem. 275, 23429-23438]. To investigate the effect on thermodynamics, the enthalpy and volume changes of charge separation in PS I in the menA and menB mutants were measured using pulsed time-resolved photoacoustics on the nanosecond and microsecond time scales. The observed thermodynamic data are the same for the menA and menB mutants. This is expected because the recruited quinone (A(P)) is the same in both mutants. The volume change of PS I from the mutants following charge separation on both time scales was -17 +/- 2 A(3), less than that of the wild type, -21 A(3). The quantum yield of charge separation was found to be slightly lower (85 +/- 9%) than that of wild-type PS I (96 +/- 10%). The observed reaction is assigned to the formation of P(700)(+)A(P)(-) from P(700)*A(P). An enthalpy change (DeltaH) of -0.69 +/- 0.07 eV was obtained for this reaction. In contrast, a larger enthalpy change -0.8 eV for the formation of P(700)(+)A(1)(-) from P(700)* and an apparent entropy change (TDeltaS, T = 25 degrees C) of -0.2 eV were obtained in wild-type PS I [Hou, H. J. M., and Mauzerall, D. (2006) J. Am. Chem. Soc. 128, 1580-1586]. Taking the free energy to be -0.70 eV in PS I of the mutants, the apparent entropy is close to zero in the mutants. Since the apparent entropy change for the overall reaction of the production of P(700)(+)F(A/B)(-) from P(700)* is very likely the same as that of the wild type, +0.35 eV, this implies that the reaction of P(700)(+)A(P)(-)F(A/B) --> P(700)(+)A(P)F(A/B)(-) in the mutants is almost completely entropy driven (DeltaG = -0.07 eV and TDeltaS = +0.40 eV). These results show that not only the kinetics but also the thermodynamics of electron transfer reactions in PS I are significantly affected by the recruitment of the foreign plastoquinone-9 into the A(1) site.

Differential effect of hydrocortisone on eosinophil and neutrophil proliferation.

Glucocorticosteroid therapy results in an increase in the number of circulating neutrophils and a decrease in the number of eosinophils. Utilizing the double layer soft agar technique, we examined the effect of physiologic to pharmacologic concentrations of hydrocortisone on the proliferation of human neutrophil progenitors and eosinophil progenitors from peripheral blood and bone marrow. When peripheral blood cultures were studied, eosinophil proliferation was inhibited in a dose-responsive fashion with 10(-8) - 10(-5) M hydrocortisone succinate, and comprised 49 +/- 4% of the colonies in control cultures and only 4 +/- 1% (P less than 0.01) at pharmacologic levels of hydrocortisone (10(-5) M). The number of neutrophil colonies, on the other hand, increased by 31% when 10(-5) M hydrocortisone was added to cultures. In order for corticosteroids to exert this effect, it was necessary to add them within 24 h of the initiation of culture. The effect of hydrocortisone on granulocyte proliferation could not be blocked by progesterone, a structurally analogous steroid. To determine whether hydrocortisone was acting directly on the progenitor cell or via an effector cell, its effect on modulating cell populations and stimulating-factor production was studied. Removal of E-rosetting cells and/or adherent cells did not affect the inhibition of eosinophil colony growth or the enhancement of neutrophil colony growth. Furthermore, addition of the potent inhibitor of T cell function, cyclosporin A, failed to affect eosinophil colony frequency, suggesting that inhibition of T cell function was an unlikely explanation for the observed hydrocortisone effect. Leukocyte conditioned media (LCM), derived from peripheral blood mononuclear cells incubated with hydrocortisone, was devoid of both neutrophil and eosinophil colony-stimulating activity, whereas a control LCM stimulated both neutrophil and eosinophil proliferation. The data suggest that the observed hydrocortisone effect on granulocyte colony formation is unlikely to be mediated by an intermediary, and that hydrocortisone acts directly on progenitor cells.

Five-year survival from breast cancer in Western Australia over a decade.

The aim of the present study was to investigate whether 5-year survival of patients with breast cancer in Western Australia has improved over time. We used a population-based study conducted in the State of Western Australia, to identify all cases of invasive breast cancer cases diagnosed in 1989, 1994 and 1999. Information on presentation, investigation and management was extracted from medical records of each case and status at 5 years after date of diagnosis was determined. Comparison of 5-year overall survival for women diagnosed in the three calendar years, and hazard ratios for survival calculated for prognostic variables were measured. We found that survival from breast cancer has improved in Western Australia since 1989. Earlier diagnosis, living in the Perth metropolitan area and use of breast-conserving surgery are associated with better survival, irrespective of year of diagnosis. Further research needs to be carried out to determine the reason for this improvement.

HER2 mRNA transcript quantitation in breast cancer.

PURPOSE: The human epidermal growth factor receptor 2 (HER2) status in breast cancer is important for prognostic prediction and the determination of optimal treatment. Current methods rely on protein expression, as determined by immunohistochemistry (IHC), as well as gene amplification as determined by in situ hybridisation (ISH). We explored whether quantitative droplet digital PCR (ddPCR) can be used for the detection and absolute quantitation of HER2 mRNA. METHODS: Digital droplet PCR (ddPCR) was performed for HER2 mRNA on 178 formalin-fixed paraffin-embedded (FFPE) breast cancer specimens. HER2 positive, equivocal and negative cases as defined by standard criteria were included and both core biopsies and tissue sections were assessed. RESULTS: HER2 positive cases contained significantly higher levels of HER2 mRNA (169-1,000,000 copies/µl) by ddPCR compared with equivocal (112-139 copies/µl, p = 0.025) and negative cases (6.2-644 copies/µl. p < 0.001). A continuum of transcript quantity was observed but a cutoff of 490 copies/µl distinguished between HER2 positive and negative cases. Results were consistent between core biopsy and tissue sections. CONCLUSIONS: ddPCR can be used to quantify HER2 mRNA transcripts in FFPE breast cancer specimens. Our results highlight the potential of ddPCR on FFPE tissue to be used to accurately quantify HER2 transcripts. Validation in large cohorts will be required to determine a clinically applicable cutoff.

Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer.

PURPOSE: Immunohistochemistry (IHC) is a newer technique for assessing the estrogen receptor (ER) status of breast cancers, with the potential to overcome many of the shortcomings associated with the traditional ligand-binding assay (LBA). The purpose of this study was to evaluate the ability of ER status determination by IHC, compared with LBA, to predict clinical outcome-especially response to adjuvant endocrine therapy-in a large number of patients with long-term clinical follow-up. PATIENTS AND METHODS: ER status was evaluated in 1,982 primary breast cancers by IHC on formalin-fixed paraffin-embedded tissue sections, using antibody 6F11 and standard methodology. Slides were scored on a scale representing the estimated proportion and intensity of positive-staining tumor cells (range, 0 to 8). Results were compared with ER values obtained by the LBA in the same tumors and to clinical outcome. RESULTS: An IHC score of greater than 2 (corresponding to as few as 1% to 10% weakly positive cells) was used to define ER positivity on the basis of a univariate cut-point analysis of all possible scores and disease-free survival (DFS) in patients receiving any adjuvant endocrine therapy. Using this definition, 71% of all tumors were determined to be ER-positive by IHC, and the level of agreement with the LBA was 86%. In multivariate analyses of patients receiving adjuvant endocrine therapy alone, ER status determined by IHC was better than that determined by the LBA at predicting improved DFS (hazard ratios/P = 0.474/.0008 and 0.707/.3214, respectively) and equivalent at predicting overall survival (0.379/.0001 and 0.381/.0003, respectively). CONCLUSION: IHC is superior to the LBA for assessing ER status in primary breast cancer because it is easier, safer, and less expensive, and has an equivalent or better ability to predict response to adjuvant endocrine therapy.

Pathology reporting of breast cancer: trends in 1989-1999, following the introduction of mammographic screening in Western Australia.

BACKGROUND: A survey of pathology reporting of breast cancer in Western Australia in 1989 highlighted the need for improvement. The current study documents (1) changes in pathology reporting from 1989 to 1999 and (2) changes in patterns of histopathological prognostic indicators for breast cancer following introduction of mammographic screening in 1989. METHODS: Data concerning all breast cancer cases reported in Western Australia in 1989, 1994 and 1999 were retrieved using the State Cancer Registry, Hospital Morbidity data system, and pathology laboratory records. RESULTS: Pathology reports improved in quality during the decade surveyed. For invasive carcinoma, tumour size was not recorded in 1.2% of pathology reports in 1999 compared with 16.1% in 1989 (p<0.001). Corresponding figures for other prognostic factors were: tumour grade 3.3% and 51.6% (p<0.001), tumour type 0.2% and 4.1% (p<0.001), vascular invasion 3.7% and 70.9% (p<0.001), and lymph node status 1.9% and 4.5% (p = 0.023). In 1999, 5.9% of reports were not in a synoptic/checklist format, whereas all reports were descriptive in 1989 (p<0.001). For the population as a whole, the proportion of invasive carcinomas <1 cm was 20.9% in 1999 compared with 14.5% in 1989 (p<0.001); for tumours <2 cm the corresponding figures were 65.4% and 59.7% (p = 0.013). In 1999, 30.5% of tumours were histologically well-differentiated compared with 10.6% in 1989 (p<0.001), and 61.7% were lymph node negative in 1999 compared with 57.1% in 1989 (p = 0.006). Pure ductal carcinoma in situ (DCIS) constituted 10.9% and 7.9% of total cases of breast carcinoma in 1999 and 1989, respectively (p = 0.01). CONCLUSIONS: Quality of pathology reporting improved markedly over the period, in parallel with adoption of standardised synoptic pathology reports. By 1999, recording of important prognostic information was almost complete. Frequency of favourable prognostic factors generally increased over time, reflecting expected effects of mammographic screening.

The Effect of Bacteriochlorophyll g Oxidation on Energy and Electron Transfer in Reaction Centers from Heliobacterium modesticaldum.

The heliobacteria are a family of strictly anaerobic, Gram-positive, photoheterotrophs in the Firmicutes. They make use of a homodimeric type I reaction center (RC) that contains ∼20 antenna bacteriochlorophyll (BChl) g molecules, a special pair of BChl g' molecules (P800), two 8(1)-OH-Chl aF molecules (A0), a [4Fe-4S] iron-sulfur cluster (FX), and a carotenoid (4,4'-diaponeurosporene). It is known that in the presence of light and oxygen BChl g is converted to a species with an absorption spectrum identical to that of Chl a. Here, we show that main product of the conversion is 8(1)-OH-Chl aF. Smaller amounts of two other oxidized Chl aF species are also produced. In the presence of light and oxygen, the kinetics of the conversion are monophasic and temperature dependent, with an activation energy of 66 ± 2 kJ mol(-1). In the presence of oxygen in the dark, the conversion occurs in two temperature-dependent kinetic phases: a slow phase followed by a fast phase with an activation energy of 53 ± 1 kJ mol(-1). The loss of BChl g' occurs at the same rate as the loss of Bchl g; hence, the special pair converts at the same rate as the antenna Chl's. However, the loss of P800 photooxidiation and flavodoxin reduction is not linear with the loss of BChl g. In anaerobic RCs, the charge recombination between P800(+) and FX(-) at 80 K is monophasic with a lifetime of 4.2 ms, but after exposure to oxygen, an additional phase with a lifetime of 0.3 ms is observed. Transient EPR data show that the line width of P800(+) increases as BChl g is converted to Chl aF and the rate of electron transfer from A0 to FX, as estimated from the net polarization generated by singlet-triplet mixing during the lifetime of P800(+)A0(-), is unchanged. The transient EPR data also show that conversion of the BChl g results in increased formation of triplet states of both BChl g and Chl aF. The nonlinear loss of P800 photooxidiation and flavodoxin reduction, the biphasic backreaction kinetics, and the increased EPR line width of P800(+) are all consistent with a model in which the BChl g'/BChl g' and BChl g'/Chl aF' special pairs are functional but the Chl aF'/Chl aF' special pair is not.

Comparison of vascularity and angiogenesis in primary invasive mammary carcinomas and in their respective axillary lymph node metastases.

It is well established that the ability of a neoplasm to induce a blood supply from a pre-existing circulation (angiogenesis) is a major factor in tumour growth, invasion and metastasis. However, the angiogenic potential of metastases and their subsequent growth have not been extensively studied. The question arises: can metastatic clones induce the same level of angiogenesis as in the primary neoplasm they emanated from? In this study it is hypothesised that in the same patient the level of vascularity and angiogenesis is the same in both the primary invasive ductal carcinoma and in the axillary lymph node metastasis at the time of surgery, according to Kerbels theory of clonal-dominance. To directly address the hypothesis, morphological measures of the established blood/lymphatic circulation (vascularity) as well as estimates of angiogenesis (endothelial cell proliferation) were measured in primary tumours and directly compared to the same parameters in the corresponding lymph node metastasis in a case by case basis (n = 17). The results demonstrate varying associations between the level of vascularity and angiogenesis between matched individual tumours and their metastatic lymph nodal deposits. It is possible that either variations in the angiogenic characteristics of the metastasising clone or local or systemic promoters or inhibitors of angiogenesis influence tumour angiogenesis at the different sites.

Inhibition of MCF-7 breast cancer cell proliferation by 5alpha-dihydrotestosterone; a role for p21(Cip1/Waf1).

Androgens inhibit the growth of breast cancer cells in vitro and in vivo by mechanisms that remain poorly defined. In this study, treatment of asynchronously growing MCF-7 breast cancer cells with the androgen, 5alpha-dihydrotestosterone (DHT), was shown to inhibit cell proliferation and induce moderate increases in the proportion of G1 phase cells. Consistent with targeting the G1-S phase transition, DHT pretreatment of MCF-7 cultures impeded the serum-induced progression of G1-arrested cells into S phase and reduced the kinase activities of cyclin-dependent kinase (Cdk)4 and Cdk2 to less than 50% of controls within 3 days. DHT treatment was associated with greater than twofold increases in the levels of the Cdk inhibitor, p27(Kip1), while p21(Cip1/Waf1) protein levels remained unchanged. During the first 24 h of DHT treatment, levels of Cdk4-associated p21(Cip1/Waf1) and p27(Kip1) were reduced coinciding with decreased levels of Cdk4-associated cyclin D3. In contrast, DHT treatment caused increased accumulation of Cdk2-associated p21(Cip1/Waf1), with no significant alterations in levels of p27(Kip1) bound to Cdk2 complexes. These findings suggest that DHT reverses the Cdk4-mediated titration of p21(Cip1/Waf1) and p27(Kip1) away from Cdk2 complexes, and that the increased association of p21(Cip1/Waf1) with Cdk2 complexes in part mediates the androgen-induced growth inhibition of breast cancer cells.

Evidence for presynaptic 5-hydroxytryptamine3 recognition sites on vagal afferent terminals in the brainstem of the ferret.

Antagonists acting at the 5-hydroxytryptamine3 receptor are potent anti-emetic agents in cases of cytotoxic- and radiation-induced vomiting, and binding sites for these compounds have been described in brainstem areas known to be involved in mediation of nausea and vomiting. We have used autoradiography to examine the distribution of one of these antagonists, [3H]granisetron in the caudal brainstem of the ferret, a commonly used animal model for physiological investigations of emesis. The highest density of binding sites was found to be in the dorsomedial region of the nucleus of the solitary tract, the principal terminus for gastric vagal afferent fibres. Lower levels of binding were observed in the area postrema and the dorsal motor nucleus of the vagus. Following unilateral nodose ganglion excision, displaceable binding of [3H]granisetron in the nucleus of the solitary tract was attenuated on the ipsilateral side by 65%. Bilateral subdiaphragmatic vagotomy abolished binding of [3H]granisetron in the entire dorsal vagal complex. These results provide strong circumstantial evidence that 5-hydroxytryptamine3 receptors are located on vagal afferent terminals in the ferret brainstem.

c-erbB-2 amplification in breast cancer: detection in formalin-fixed, paraffin-embedded tissue by in situ hybridization.

We describe a sensitive and practical in situ hybridization method, using a digoxigenin-labeled probe, for the detection of c-erbB-2 amplification in breast cancer in formalin-fixed, paraffin-embedded tissue sections. Forty-six primary breast carcinomas were studied. Nuclear hybridization signal was observed in 36 of 46 carcinomas. Signal was confined to malignant cells. Normal breast epithelium and stromal and inflammatory cells were uniformally negative. DNase predigestion, no-probe preparations, and competitive hybridization confirmed the specificity of the reaction. The hybridization reaction was localized to multiple discrete foci in tumor cell nuclei, suggesting multiple sites of gene copy and transcriptional activity in the nucleus. Considerable cell-to-cell variation in hybridization signal was evident within individual tumors and positive reactions were observed in several cases in which amplification could not be detected by either Southern or slot blot analysis. The high sensitivity and specificity of the reaction and its use in a tissue-based system will allow the study of a range of possible precursor lesions of breast cancer for evidence of c-erbB-2 amplification.