First Report of Necrotic Leaf Spot Caused by Plectosphaerella cucumerina on Lamb's Lettuce in Southern Italy.

During spring 2013, lamb's lettuce plants (Valerianella locusta) cv. Calarasi (Rijk Zwaan) in a commercial greenhouse in Sele Valley (Salerno Province, southern Italy) exhibited small, black-brown, irregular spots (1 mm2) that became necrotic, enlarged, and coalesced. The spots were mostly on the tips of leaves, and were surrounded by a yellow halo. The disease was severe under greenhouse conditions of 60 to 90% RH and maximum air temperature of 26°C, and affected up to 70% of the plants. The greenhouses covered an area of ~3,000 ha where many salad species are grown. Tissue fragments were excised from symptomatic leaves, sterilized by sequential dipping in 70% ethanol for 30 s and in 1% NaOCl for 30 s, rinsed in sterilized distilled water, and placed in 9-cm-diameter petri dishes containing potato dextrose agar (PDA) medium amended with streptomycin sulfate (0.1 g/liter). The plates were incubated at 24°C in the dark. A fungus was isolated consistently from infected leaf tissue after 4 days. Each colony was whitish to orange. Mycelium was hyaline, branched, septate, 3 to 4 µm wide, with numerous anastomosis-forming hyphal coils. Conidiophores were solitary, hyaline, smooth, thin-walled, unbranched or rarely irregularly branched. Conidiogenous cells were phialidic, determinate, discrete, smooth, solitary, and formed on hyphal coils. Phialides were aseptate or occasionally 1-septate near the base. Conidia (n = 100) were ellipsoidal, hyaline, smooth, septate or aseptate, and 6.6 ± 0.9 × 2.8 ± 0.4 µm. On the basis of morphological criteria (3), the fungus was ascribed to Plectosphaerella cucumerina (L.) Laterr. (anamorph Plectosporium tabacinum). An aliquot (50 ng) of genomic DNA extracted from mycelium of five cultures obtained by monosporic isolation on PDA was used as template for a PCR reaction with primers ITS5/ITS4, specific for the ITS 5.8S rDNA region of fungi (3). The 500-bp sequences amplified from the five isolates were identical, and the sequence of isolate Val-2 was submitted to GenBank (KF753234). Sequence analysis with BLASTn showed 100% identity of this sequence to the ITS-5.8S rDNA sequences of 11 isolates of P. cucumerina in GenBank. Three isolates were selected for pathogenicity tests on the lamb's lettuce cv. Calarasi. Before planting, seeds were surface-disinfected in 1% NaOCl and rinsed with sterilized distilled water. Plants (35 days old, 30 plants tested/isolate) were grown in 0.7-liter pots filled with a sterilized (autoclaved at 112°C for 1 h on each of two consecutive days) mixture of soil:sand:perlite (2:1:1), and inoculated by spraying the leaves with a spore suspension (106 CFU/ml, ~3 ml applied/plant) of each isolate prepared from 7-day-old cultures on PDA. As a control, five plants were sprayed with sterilized water. All plants were incubated in a growth chamber at 90% RH with a 12-h photoperiod at 24°C. Leaf spots typical of those on the original symptomatic plants appeared 7 to 10 days after inoculation on all inoculated plants. No symptoms were observed on control plants. P. cucumerina was re-isolated only from symptomatic leaves, as described for the original isolations. P. cucumerina has been associated with root and collar roots of some horticultural crops (1), and a leaf spot on Diplotaxis tenuifolia (2), often grown in rotation with lamb's lettuce in southern Italy. To our knowledge, this is the first report of P. cucumerina as a pathogen of V. locusta in Italy or elsewhere. The disease caused economic loss to lamb's lettuce, primarily used in Italy in fresh-cut, mixed salads. References: (1) A. Carlucci et al. Persoonia 28:34, 2012. (2) A. Garibaldi et al. Plant Dis. 96:1825, 2012. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

New report of Phoma glomerata on Coriandrum sativum L.

In 2011, coriander plants (Coriandrum sativum L.), showing symptoms of greyish to hell brown lesions with dark brown border between discoloured and asymptomatic tissues, were observed in Bulgaria. Pycnidia with small unicellular conidia appeared in the pale centre of the lesions. In this work, the identification of the causal agent of this disease was made applying Koch's postulates. Moreover, for a rapid and unambiguous identification of the fungal species, the internal transcribed spacers and the 5.8 rDNA gene (ITS1-5.8-ITS2) were amplified by PCR from DNA extracted from the isolated colonies. The amplicon was sequenced and analyzed using BLASTn, and showed a homology of 100% with a corresponding sequence of Phoma glomerata (accession number DQ093699). The fungus isolated, after the morphological and molecular characterization, was ascribed to the species Phoma glomerata (Corda) Wollenweber and Hochapfel. This is the first report of P. glomerata as agent of stem rot of coriander in Bulgaria and elsewhere.

Morphological and molecular characterisation of fungal populations possibly involved in the biological alteration of stones in historical buildings.

The deterioration process of historical building is progressive and irreversible, and the timing and mode of impact are different depending on the characteristics of building materials used, local microclimate, air pollution, presence of specific flora and fauna. The chemical and microbiological characterisation of building materials is mandatory in preventing and eventually recovering degradation effects. Ideally, the analysis of structural stones should be complete, efficient, rapid, and non destructive when dealing with a precious or unique construction. The investigation has been performed on a private historical building made using calcarenite stones and sited between the archaeological site of Lavello, a little town located in the Basilicata Region (South Italy), and the industrial area surrounding this town. To study in progress the degradation of stone materials, a new building sample (ca. 1 m3) was constructed by using the same stones (33 x 15cm), collected from a local quarry. The intact calcarenite stone was characterised by using different methods of surface analysis (XRD, XPS, SEM), and exposed to outdoor conditions. The analyses of the stone material were repeated after three and six months to early evaluate the progression of alterations and the forward modifications of calcarenite structure. After only three months of the new building sample exposure, the adopted analytical methods were able to provide a series of data, which allowed the assessment of the incipient modification of the stone surfaces. The degradation appeared worsened performing the same observations on sixth month replicates, suggesting that environmental conditions modified the structure and the compactness of stones and favoured the biological colonization of surfaces especially in the South-East direction of prevailing winds. For this reason the presence of fungi on the stones' surface was investigated and a morphological and molecular characterization of sampled fungi was performed. Several genera and species of fungi, possibly, involved in degradation were found. The most frequent colonies belonged to Alternaria (A. infectoria, A. citri and Alternaria sp.), Coprinopsis sp., Penicillium piceum, Fusatrium equiseti and Scytalidium termophilus.

First Report of Tomato spotted wilt virus on Tobacco in Campania, Italy.

During the spring and summer of 2010, a survey for viruses was conducted in two tobacco field trials at the Agricultural Research and Experimentation Council in Scafati, Campania, Italy. A total of 1,392 symptomatic and asymptomatic tobacco plants (cv. Burley) were sampled, and leaves were analyzed by double-antibody sandwich-ELISA using polyclonal antisera against five tobacco-infecting viruses: Tobacco mosaic virus, Potato virus Y, Cucumber mosaic virus, Alfalfa mosaic virus, and Tomato spotted wilt virus (TSWV) (Loewe, Munich, Germany). Only one plant was positive to TSWV. Symptoms on this plant were severe necroses on the stem with a few chlorotic/necrotic leaflets on the top of the plant. This result was subsequently confirmed by reverse transcription (RT)-PCR. The primers (5'-ATGTCTAAGGTTAAGCTC-3' forward and 5'-TTAAGCAAGTTCTGTGAG-3' reverse) targeted the nucleocapsid gene of TSWV and amplified the expected product of approximately 800 bp (2). The resultant sequence (GenBank Accession No. JF290419) was aligned and edited using BlastN, displaying 99.9% identity with deposited TSWV nucleocapsid gene sequences in GenBank, with no similarity to any other targets, which confirmed the presence of TSWV in tobacco. Leaf homogenate from the tobacco symptomatic plants was inoculated onto three plants of Nicotiana benthamiana, N. glutinosa, and Datura metel. All plants developed a systemic necrosis after 7 days. Inoculation from N. glutinosa back to cv. White Burley tobacco produced symptoms similar to those observed in the field. Two plants from each species were used as noninoculated controls, which remained asymptomatic. TSWV infection has been responsible for severe epidemics on tobacco throughout the United States and Greece, with losses estimated as much as 85% (1,3). The presence of TSWV in Italy could therefore represent a serious threat for tobacco in the region, especially considering that it is prevalent in other crops in the area and vectors are widespread. References: (1) E. K. Chatzivassiliou. Plant Dis. 92:1012, 2008. (2) R. K. Jain et al. Plant Dis. 82:900, 1998. (3) B. Mandal et al. Ann. Appl. Biol. 151:67, 2007.

First Report of Crown and Root Rot Caused by Rhizoctonia solani AG-4 on Orange Jessamine in Italy.

Murraya paniculata (L.) Jack, commonly called orange jessamine or orange jasmine (Rutaceae), is a small tropical tree that is native to Asia. This species, closely related to Citrus, is grown as an ornamental tree or hedge. During October of 2007, crown and root rot was observed on approximately 12,000 pot-grown, 4-month-old plants in a nursery in eastern Sicily, Italy. Basal leaves turned yellow and gradually became necrotic, and infected plants often died. Disease symptoms were observed on 1,800 (15%) plants. Isolations from affected tissues on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/liter recovered a fungus with mycelial and morphological characteristics consistent with Rhizoctonia solani Kühn. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Microscopic examination revealed that hyphae had a right-angle branching pattern, were constricted at the base of the branch near the union with main hyphae, and were septate near the constriction. The nuclear condition of hyphal cells was determined on cultures grown at 25°C on 2% water agar (WA) when stained with 3% safranin O solution and examined at ×400. Anastomosis groups were determined by pairing isolates on 2% WA in petri plates (4). Pairings were made with tester strains AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11. Anastomosis was observed only with tester isolates of AG-4 producing both C2 and C3 reactions. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. These results were consistent with other reports on anastomosis reactions (1). The identification of group AG-4 within R. solani has been confirmed by electrophoretic patterns of pectic enzymes (polygalacturonases) in vertical pectin-acrylamide gel stained with ruthenium red (2). Pathogenicity tests were conducted on potted, healthy, 6-month-old seedlings of orange jessamine. Twenty-five plants were inoculated by placing 1-cm2 PDA plugs from 5-day-old mycelial cultures near the base of the stem. The same number of plants inoculated with PDA plugs served as controls. Plants were maintained at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Wilt symptoms, identical to ones observed in the nursery, developed 3 months after inoculation because of crown and root rot. Control plants remained disease free. The pathogen was reisolated from symptomatic tissues, completing Koch's postulates. Collar rot due to R. solani was previously detected on M. koenigii (3). To our knowledge, this is the first report of R. solani causing disease on M. paniculata. References: (1) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (2) R. H. Cruickshank and G. C. Wade. Anal. Biochem. 107:177, 1980. (3) A. C. Jain and K. A. Mahmud. Rev. Appl. Mycol. 32:460, 1953. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Crown Rot, Stem Rot, and Root Rot Caused by Binucleate Rhizoctonia AG-G on Viburnum tinus in Italy.

Laurustinus (Viburnum tinus L.), native to the Mediterranean Region, is an evergreen shrub belonging to the Caprifoliaceae that is commonly cultured as an ornamental shrub or small tree. During the summer and autumn of 2007 and 2008, a widespread yellowing, partial foliar necrosis, or death of the whole plant was observed on 3- to 4-year-old potted plants of V. tinus in a commercial nursery in eastern Sicily (Italy). More than 20% of the plants showed disease symptoms. Infected roots, crowns, and stems turned dark brown, leaves gradually became necrotic, and infected plants were often killed. Diseased tissues were disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/liter, and then incubated at 25°C. A binucleate Rhizoctonia (BNR) species was consistently isolated from affected tissue of Laurustinus. Fungal colonies were initially white, then turned light brown or brown with age, and formed irregularly shaped, light brown sclerotia after 10 days. Hyphal cells were determined to be binucleate when stained with 1% safranin O and 3% KOH solution (1) and examined with a microscope at ×400. Anastomosis groups were determined by pairing isolates on 2% water agar in petri plates (4). Pairings were made with tester strains of binucleate Rhizoctonia AG-A through AG-S, except AG-J and AG-M. Anastomosis was observed only with tester isolates of AG-G. The rDNA-ITS of two isolates of BNR (DISTEF-Vt 31 and DISTEF-Vt 32) was sequenced (GenBank Accession Nos. AB478783 and AB478784, respectively) (3). The sequence from these two isolates exhibited 100% homology with BNR AG-G (GenBank Accession No. AY927334). Pathogenicity tests were conducted on potted, healthy, 6-month-old laurustinus. Twenty plants were inoculated by placing 1-cm2 plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1-cm2 PDA plugs as control. Plants were kept at 25°C and 95% relative humidity with a 12-h fluorescent light/dark regimen. Stem, crown, and root rot symptoms, identical to ones observed in nursery, appeared 20 days after inoculation, and all the inoculated plants showed symptoms within 1 month. Control plants remained healthy. Binucleate Rhizoctonia was reisolated from symptomatic tissues, completing Koch's postulates. R. solani was previously reported on Viburnum sp. in the United States (2). To our knowledge, this is the first report of binucleate Rhizoctonia causing disease on V. tinus. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. F. Farr et al. Page 1252 in: Fungi on Plants and Plant Products in the United States. The American Phytopathological Society. St. Paul, MN, 1989. (3) M. Hyakumachi et al. Phytopathology 95:784, 2005. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Damping-Off on African Daisy Caused by Rhizoctonia solani AG-4 in Italy.

Osteospermum (African Daisy or Cape Daisy) is a genus belonging to the Calendulae with a large number of perennial plant species. In February of 2007, a severe damping-off occurred on 3- to 4-month-old potted cuttings of Osteospermum 'Impassion Rose Purple', 'Impassion White', 'Impassion Purple', and 'Impassion White Rose' cultivated in a nursery in eastern Sicily. More than 30% of the plants were infected. Disease symptoms consisted of extensive water-soaked lesions at the base of the stem followed by wilt and collapse of the plant. Isolations from diseased tissues on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/l consistently recovered a fungus with morphological characteristics of Rhizoctonia solani. Fungal colonies were initially white, turned brown after 2 to 3 days, and produced irregularly shaped, brown sclerotia after 1 week. Microscopic examination revealed that hyphae had right angle branching patterns, were constricted at the base of the branch near the union with main hyphae, and septate near the constriction. The number of nuclei per hyphal cell was determined on cultures grown at 25°C on 2% water agar in petri plates. Mycelium was stained with 0.5% aniline blue solution (4) and examined with a microscope at ×400. The hyphal cells were all multinucleate. Anastomosis groups were determined by pairing isolates (3) with tester isolates of AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11. Anastomosis was observed only with tester isolates of AG-4, giving C2 reactions (1) at a high frequency. The identification of group AG-4 within R. solani had been obtained by electrophoretic patterns of pectic enzymes (polygalacturonases) in vertical pectin-acrylamide gel stained with ruthenium red (2). All isolates of R. solani collected from infected plants were paired in all combinations on PDA plus 1% activated charcoal and examined for somatic interaction. All paired colonies merged without producing visible tufts of aerial mycelium. Absence of tufts and the lack of formation of heterokaryon at the hyphal interaction zone indicated that all isolates belonged to the same mating type with the same mating alleles (3). Pathogenicity tests were performed by placing plugs of PDA from 5-day-old mycelial cultures in the soil near the base of the stem on 20 potted, healthy, 2-month-old cuttings of Osteospermum cv. Impassion Rose Purple. The same number of plants treated with 1/cm2 PDA plugs served as controls. Following inoculation, all plants were maintained in a growth chamber at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Wilt symptoms and lesions at the base of stem identical to those observed in the nursery developed 7 days after inoculation, and all inoculated plants died within 20 days. Control plants remained symptomless. R. solani AG-4 was consistently reisolated from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report of damping-off on the genus Osteospermum caused by R. solani. References: (1) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (2) R. H. Cruickshank and G. C. Wade. Anal. Biochem. 107:177, 1980. (3) M. C. Juliàn et al. Phytopathology 86:566, 1996. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Damping-Off Caused by Rhizoctonia solani AG-4 on Lagunaria patersonii in Italy.

Lagunaria patersonii (Adr.) G. Don (cow itch tree) is native to Australia and tolerates salted winds. During July 2007, damping-off of cow itch tree was observed on 4-month-old seedlings growing in a commercial nursery in eastern Sicily, Italy. More than 20% of the seedlings showed disease symptoms. First symptoms consisting of water-soaked lesions at the seedling base that expand rapidly girdle the stem and collapse the seedling in a few days. Diseased tissues were disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with streptomycin sulphate at 100 mg/l, and then incubated at 25°C. A fungus with mycelial and morphological characteristics of Rhizoctonia solani Kühn was consistently yielded. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Microscopic examination revealed that hyphae had a right-angle branching pattern, were constricted at the base of the branch near the union with main hyphae, and septate near the constriction. Basidia were not observed in the greenhouses or on the plates. Hyphal cells were determined to be multinucleate when stained with 0.5% aniline blue solution and examined at ×400 magnification with a microscope. Anastomosis groups were determined by pairing isolates on 2% water agar in petri plates (3). Pairings were made with tester strains of AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, AG-11. Anastomosis was observed only with tester isolates of AG-4 producing both C2 and C3 reactions. The hyphal diameter at the point of anastomosis was reduced, the anastomosis point was obvious, and cell death of adjacent cells was observed. These results were consistent with other reports on anastomosis reactions (1). The identification of group AG-4 within R. solani has been confirmed by electrophoretic patterns of pectic enzymes (polygalacturonases) in vertical pectin-acrylamide gel stained with ruthenium red (2). Pathogenicity tests were conducted on potted, healthy, 3-month-old seedlings of cow itch tree. Twenty plants were inoculated by placing plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1 cm2 PDA plugs as control. Plants were kept at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Wilt symptoms due to basal stem rot, identical to ones observed in the nursery, appeared 10 days after inoculation and all inoculated plants showed symptoms within 1 month. Control plants remained healthy. The pathogen was reisolated from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report in the world of R. solani causing disease on L. patersonii. References: (1) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (2) R. H. Cruickshank and G. C. Wade. Anal. Biochem. 107:177, 1980. (3) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Sclerotinia sclerotiorum on Buckwheat (Fagopyrum esculentum) in Italy.

In each of two fields of buckwheat (Fagopyrum esculentum L.) grown in Benevento Province (southern Italy), 60 to 70% of the plants developed severe root and crown rot. Symptoms included irregular, water-soaked spots on stems that were eventually covered with cottony mycelia as the lesions enlarged. Black sclerotia usually developed within the mycelium. The fungus was isolated on potato dextrose agar and 2% water agar. On the basis of colony morphology, including the production of black sclerotia (1), the fungus was identified as Sclerotinia sclerotiorum (Lib.) De Bary. The identity of the fungus was confirmed by near exact identity of internal transcribed spacer sequences (99%) with two isolates of S. sclerotiorum in GenBank (Accession Nos. Z73800 and Z73799). Pathogenicity of the fungus on buckwheat was evaluated by transplanting 20 20-day-old healthy plants in a mixture of soil and fungal inoculum (0.5% of wet millet seeds colonized by four isolates of S. sclerotiorum). Lesions on crowns and roots developed after 12 days and sclerotia appeared approximately 20 days later. No symptoms developed on noninoculated plants. Reisolation from inoculated plants yielded colonies of S. sclerotiorum. To our knowledge, this is the first report of S. sclerotiorum on buckwheat in Italy. The high incidence and severity of the disease may be limiting factors in the development of buckwheat as an alternative crop of tobacco in southern Italy. Reference: (1) J. E. M. Mordue and P. Holliday. Sclerotinia sclerotiorum. No. 513 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1976.

Cyclin D- and E-dependent kinases and the p57(KIP2) inhibitor: cooperative interactions in vivo.

This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.

Inhibition of ras-induced proliferation and cellular transformation by p16INK4.

The cyclin-dependent kinase 4 (CDK4) regulates progression through the G1 phase of the cell cycle. The activity of CDK4 is controlled by the opposing effects of the D-type cyclin, an activating subunit, and p16INK4, an inhibitory subunit. Ectopic expression of p16INK4 blocked entry into S phase of the cell cycle induced by oncogenic Ha-Ras, and this block was relieved by coexpression of a catalytically inactive CDK4 mutant. Expression of p16INK4 suppressed cellular transformation of primary rat embryo fibroblasts by oncogenic Ha-Ras and Myc, but not by Ha-Ras and E1a. Together, these observations provide direct evidence that p16INK4 can inhibit cell growth.

Suppression of Myc, but not E1a, transformation activity by Max-associated proteins, Mad and Mxi1.

Mad and Mxi1, two members of the Myc-related basic-region helix-loop-helix/leucine-zipper family of proteins, associate directly with Max to form sequence-specific DNA binding heterodimers that are transactivation-incompetent. Mad-Max complexes have been shown to exert a strong repressive effect on Myc-induced transactivation, perhaps through the competitive occupation of common promoter binding sites also recognized by active Myc-Max heterodimers. To place these recent biochemical observations in a biological context, mad and mxi1 expression vectors were tested for their ability to influence Myc transformation activity in the rat embryo fibroblast cooperation assay. Addition of an equimolar amount of mad or mxi1 expression vector to mouse c-myc/ras cotransfections resulted in a dramatic reduction in both the number of foci generated and the severity of the malignant phenotype. Myc-specific suppression by Mad and Mxi1 was demonstrated by their ability to affect c- and N-myc-, but not ela-, induced transformation. In contrast, mad and mxi1 expression constructs bearing deletions in the basic region exerted only mild repressive effects on Myc transformation activity, suggesting that occupation of common DNA binding sites by transactivation-incompetent Mad-Max or Mxi1-Max complexes appears to play a more dominant role in this suppression than titration of limited intracellular pools of Max away from active Myc-Max complexes. Thus, these biological data support a current model for regulation of Myc function in which relative intracellular levels of Mad and Mxi1 in comparison to those of Myc may determine the degree of activation of Myc-responsive growth pathways.

Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under control of the rabbit uteroglobin promoter in transgenic mice.

Transgenic mice bearing two fragments of the rabbit uteroglobin 5'-flanking region fused to the new reporter gene (pac) encoding puromycin N-acetyltransferase (PAC) showed a different pattern of expression. Transgenic lines (C0.4) harboring a 404-bp fragment (-396/+8) had a uterus-specific expression slightly inducible by estrogen, lacking detectable expression in other tissues where the uteroglobin-encoding gene is naturally expressed in rabbit. Transgenic lines (C3.2) bearing a longer fragment of 3.2-kb (-3254/+8) showed hormonally regulated expression in the uterus and the male genital tract, and detectable expression in the lung. In addition, the nonstimulated uterine expression of the transgene was higher in C0.4 lines than in C3.2 lines. It could be concluded that all sequences required for uterus-specific expression should be present within the 404-bp fragment, and that other upstream (-396) sequences are responsible for expression in the lung and male genital tract, as well as for a possible down modulation of expression in the uterus.

Immunocytochemical localization of phospholipase A2 in hamster spermatozoa.

Antibodies raised against porcine pancreatic phospholipase A2 (PLA2) react in immunoblottings with both the antigen as well as with one protein band of about 14 kDa from hamster spermatozoa extracts. Immunoblottings of proteins extracted from spermatozoon head and tail fractions also show similar results. Anti-PLA2 purified IgGs were employed for light and electron microscopic immunocytochemistry in order to detect PLA2 in hamster cauda epididymal spermatozoa. When whole mount spread spermatozoa were used under light (employing the PAP complex) or electron microscopy (using anti-rabbit gold conjugated), the acrosomal area of the gametes shows a noticeable labelling; a characteristic which is not observed in samples treated with the pre-immune serum. Immunocytochemistry undertaken in ultrathin sections from spermatozoon samples embedded in Lowicryl, demonstrates that the antigen appears preferentially distributed in the acrosome. Besides, sperm tails showed a scattered distribution of gold granules in the mitochondria of the midpiece. Results suggest that the antibody used recognizes a PLA2 which is preferentially located in the acrosome and mitochondria. On the other hand, the presence of a surface PLA2 in the plasma membrane covering the acrosome is suggested. This surface PLA2 would be probably related to the acrosome reaction phenomenon that occurs in the spermatozoon before penetrating the oocyte.