V3 Protein Research Articles

Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation

Variants of the oncogenic EML4-ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4-ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4-ALK V3 but not V1 protein re-localises to microtubules, an effect recapitulated in a catalytically inactive EML4-ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild-type and catalytically inactive EML4-ALK V3 proteins, but not a Lys-Glu salt bridge mutant. We propose that EML4-ALK foci formation occurs as a result of transient association of stable EML4-ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4-ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.

Geminiviruses encode additional small proteins with specific subcellular localizations and virulence function

Geminiviruses are plant viruses with limited coding capacity. Geminivirus-encoded proteins are traditionally identified by applying a 10-kDa arbitrary threshold; however, it is increasingly clear that small proteins play relevant roles in biological systems, which calls for the reconsideration of this criterion. Here, we show that geminiviral genomes contain additional ORFs. Using tomato yellow leaf curl virus, we demonstrate that some of these small ORFs are expressed during the infection, and that the encoded proteins display specific subcellular localizations. We prove that the largest of these additional ORFs, which we name V3, is required for full viral infection, and that the V3 protein localizes in the Golgi apparatus and functions as an RNA silencing suppressor. These results imply that the repertoire of geminiviral proteins can be expanded, and that getting a comprehensive overview of the molecular plant-geminivirus interactions will require the detailed study of small ORFs so far neglected.

Specific synonymous mutations tightly correlate with HIV-1 co-receptor usage and differentially affect the secondary structure of HIV-1 Env RNA.

Human immunodeficiency virus (HIV) is a pathogen that infects blood cells, using CD4 molecule and two cell receptors CCR5 and CXCR4. The other major actor is gp120/gp41 viral protein complex, which interacts with receptors. Here, the presence of synonymous mutations associated with HIV-1 tropism and the related RNA secondary-structure in HIV-1 infected patients was evaluated. The analysis includes gp120-sequences from 340 HIV-1 subtype-B infected patients, all retrieved from Los Alamos database and with phenotypic HIV tropism determination based on recombinant-virus entry-assay. Frequencies of all nucleotide substitutions were calculated. Mfold and RNAfold algorithms were used to predict RNA secondary-structure of HIV-1. Nineteen codons in V2/C2, V3 and C3 domains were found to be closely related to CCR5 and CXCR4. Additionally, in X4-sequences, gp120 gca303gcu and gua222guc synonymous mutations are positively related to the gp120 S11R and T8A/I codons in V3 protein domain. Furthermore, gua222guc increases stability of the viral RNA secondary-structure. Probably, it would not be surprising if a novel escape viral strategy therapy will be related to the gp120 synonymous mutations. Moreover, in relation to the pivotal role played by gp120 in polyvalent vaccine approaches, the impact of gp120 synonymous mutations may play an important role in HIV entry into the cell. Keywords: gp120; tropism; v3; s11r; evolution; vaccine.

Production and Evaluation of the Properties of HIV-1-Nef-MPER-V3 Fusion Protein Harboring IMT-P8 Cell Penetrating Peptide.

Finding a safe and effective vaccine for HIV-1 infection is still a major concern. This study aimed to design and produce a recombinant Nef-MPER V3 protein fused with IMT-P8 using E. coli expression system to provide a potential HIV vaccine with high cellular penetrance. After synthesizing the DNA sequence of the fusion protein, the construct was inserted into the pET-28 expression vector. The recombinant protein expression was induced using 1 mM IPTG and the product was purified through affinity chromatography. Characterization of cellular delivery, toxicity and immunogenicity of the protein was carried out. The recombinant protein was expressed and confirmed by the anti-Nef antibody through western blotting. Data analyses showed that the protein possessed no considerable toxicity effect and has improved the IMT-P8 penetration rate in comparison to a control sample. Moreover, the antigen immunogenicity of the protein induced specific humoral response in mice. It was concluded that IMT-P8-Nef-MPER-V3 fusion protein has a high penetrance rate in mammalian cell line and low toxicity, thus it can be potentially considered as a vaccine against HIV-1.

Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease.

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

V3 protein encoded by mulberry crinkle leaf virus acts as a pathogenicity determinant in Nicotiana benthamiana

A potential pathogenicity determinant and its critical amino acid (aa) sequences of mulberry crinkle leaf virus (MCLV) were determined through expression of MCLV genes in Nicotiana benthamiana using a potato virus X (PVX) vector. Among six genes encoded by MCLV, expression of only V3 resulted in a severe downward-leaf-curling symptom in newly emerging leaves of N. benthamiana plants, which suggested that the V3 protein is a pathogenicity determinant of MCLV. Analyses of truncation mutations indicated that the 94 residues at the N terminus of V3 are sufficient to maintain the function of the pathogenicity determinant V3 in N. benthamiana.

The synthesis and secretion of versican isoform V3 by mammalian cells: A role for N-linked glycosylation

Versican is a large extracellular matrix (ECM) chondroitin sulfate (CS) proteoglycan found in most soft tissues, which is encoded by the VCAN gene. At least four major isoforms (V0, V1, V2, and V3) are generated via alternative splicing. The isoforms of versican are expressed and accumulate in various tissues during development and disease, where they contribute to ECM structure, cell growth and migration, and immune regulation, among their many functions. While several studies have identified the mRNA transcript for the V3 isoform in a number of tissues, little is known about the synthesis, secretion, and targeting of the V3 protein. In this study, we used lentiviral generation of doxycycline-inducible rat V3 with a C-terminal tag in stable NIH 3T3 cell lines and demonstrated that V3 is processed through the classical secretory pathway. We further show that N-linked glycosylation is required for efficient secretion and solubility of the protein. By site-directed mutagenesis, we identified amino acids 57 and 330 as the active N-linked glycosylation sites on V3 when expressed in this cell type. Furthermore, exon deletion constructs of V3 revealed that exons 11–13, which code for portions of the carboxy region of the protein (G3 domain), are essential for V3 processing and secretion. Once secreted, the V3 protein associates with hyaluronan along the cell surface and within the surrounding ECM. These results establish critical parameters for the processing, solubility, and targeting of the V3 isoform by mammalian cells and establishes a role for V3 in the organization of hyaluronan.

Identification and subcellular location of an RNA silencing suppressor encoded by mulberry crinkle leaf virus

Mulberry crinkle leaf virus (MCLV) is a novel geminivirus recently identified from the woody plant mulberry (Morus alba L.). Little is known about the functions of the proteins encoded by the MCLV genome. Here, all the MCLV-encoded proteins were examined for the ability to suppress gene silencing by an agroinfiltration assay in combination with northern blot analysis of green fluorescent protein (GFP) mRNA and western blot analysis. Of the six proteins, only one protein, V3, which has been predicted to play a role in viral movement, was found to suppress the gene silencing induced by a sense GFP gene in Nicotiana benthamiana 16c. The minimal amino acid sequence of V3 that maintains suppressor activity was also determined by constructing truncated mutants lacking different lengths of the amino acid sequences at the N- or C-terminus of the V3 protein. The results showed that the 94 N-terminal amino acid residues of V3 are sufficient to maintain V3 suppressor activity. In addition, the subcellular location of the V3 protein was investigated by confocal laser scanning microscopy after the expression of a V3-RFP fused protein in leaf epidermal cells of N. benthamiana. The results indicated that the V3 protein localized not only to the cytoplasm but also to the nucleus of N. benthamiana, implying that V3 can shuttle between the nucleus and the cytoplasm. Deletion mutant analysis indicated that a putative nuclear localization signal (NLS) between aa 118–134 might be responsible for the nuclear distribution of the V3 protein. Given the importance of RNA silencing in plant-virus interactions, the identification of a silencing suppressor of MCLV should be valuable in understanding the pathogenicity and molecular biology of this virus.

Investigation of the relative biological effectiveness and uniform isobiological killing effects of irradiation with a clinical carbon SOBP beam on DNA repair deficient CHO cells.

Spread-out Bragg peak (SOBP) C ions have been used clinically in charged particle radiation therapy for years. An SOBP beam consists of various monoenergetic Bragg peaks; however, the biological effect of irradiation with an SOBP beam track has not been well-studied. In order to determine the clinically prospective molecular targets, radiosensitivity to the beam track in DNA repair deficient cell lines was investigated. A total of four distinct Chinese hamster ovary (CHO) cell lines, including CHO10B2 (wild-type), V3 (protein kinase DNA-activated catalytic polypeptide deficient), 51D1 (RAD51 paralog D deficient) and PADR9 [poly(ADP-ribose) polymerase (PARP) deficient], were irradiated with gamma-rays, C ions (290 MeV/n) and Fe ions (500 MeV/n), in order to compare cellular lethality. An OptiCell™ culture system was used to evaluate the lethality at distinct depths of SOBP C ions. Relative biological effectiveness (RBE) values of C ions (linear energy transfer (LET), 13 and 70 keV/µm) and Fe ions (LET, 200 keV/µm) were calculated from cell survival using colony formation assay with standard cell dishes. All cell lines exhibited similar sensitivity to 70 keV/µm C ions and 200 keV/µm Fe ions. Furthermore, V3 cells did not exhibit increased sensitivity to high LET C ions and Fe ions, compared with low LET gamma-rays and C ions, and 51D1 cells irradiated with 13 keV/µm C ions exhibited relatively high RBE values among the tested cell lines. Conversely, PADR9 cells exhibited low RBE values for 13 keV/µm C ions and high RBE values for 70 keV/µm C ions. Obtained using the OptiCell system, the survival fractions in the SOBP region were uniform for wild-type and PADR9 cells. Conversely, V3 and 51D1 cells exhibited decreased cell death in the distal region of the SOBP. These results indicated that PARP is a more effective target for clinical beam therapy, compared with the non-homologous end joining repair and homologous recombination repair pathways. PARP deficiency may be an optimal target for C ion therapy and the results of the present study may contribute to the development of a more effective heavy ion radiation therapy.

Subcellular localization of grapevine red blotch-associated virus ORFs V2 and V3.

Grapevine red blotch-associated virus is a recently discovered plant monopartite gemini-like virus found in North American grapevines. Leaf discoloration and a decrease in fruit quality are associated with its infection. Two of its six open reading frames (ORFs), V2 and V3, are of unknown function and share no obvious homology with plant or viral genes. Transient expression of these ORFs in fusion with the green fluorescent protein demonstrated that the V2 protein localizes in the nucleoplasm, Cajal bodies, and cytoplasm; and the V3 protein localizes in various unidentified subnuclear bodies. Additionally, the V2 protein is redirected to the nucleolus upon co-expression with the nucleolus and Cajal body-associated protein Fib2.

Design and Evaluation of Meningococcal Vaccines through Structure-Based Modification of Host and Pathogen Molecules

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.

Versican G1 Domain and V3 Isoform Overexpression Results in Increased Chondrogenesis in the Developing Chick Limb In Ovo

Previous work has shown that versican proteoglycan is highly expressed in the extracellular matrix of precartilage limb mesenchyme. Although much of versican's role in chondrogenesis has been attributed to its glycosaminoglycan complement, N- and C-terminal G1 and G3 domains of versican have been shown to possess distinct functions when expressed ectopically. This study was undertaken to test the hypothesis that overexpression of the versican G1 domain and short V3 isoform, comprised of only G1 and G3, in the chick wing in ovo would result in increased chondrogenesis, suggesting function for discrete versican domains in limb skeletal development. Recombinant adenoviruses encoding G1 and V3 proteins were microinjected into proximal HH19-25 chick wing buds which resulted in significant enlargement of humeral primordia at HH35. Enhanced cartilage deposition appeared due to increased chondrogenic aggregation as a result of recombinant G1 or V3 overexpression, further implicating versican in early stages of limb development.

AN ADAPTIVE WALK BY HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 THROUGH A FLUCTUATING FITNESS LANDSCAPE

The mutational landscape model of adaptive sequence evolution has been used to explain an unexpected strong positive linear relationship between marginal fitness and mean site-specific amino acid frequency in the functionally important HIV-1 gp120 V3 protein region. The model predicts a positive linear relationship between the probability that a particular beneficial allele, among several, is the next to spread to fixation during an adaptive walk, its transition probability, and the allele's selection coefficient. Here, stochastic simulation is used to confirm the intuition that the linear relationship between transition probabilities and selection coefficients, predicted by the model, should, under fluctuating selection, produce a linear relationship between allele frequency, averaged across populations, and fitness. In addition, these relationships hold for the effective population size and mutation rate of HIV-1 and for the moderately strong selection observed for V3. A survey of the strength of mutation for diverse organisms suggests that these relationships may be widely applicable.

G-protein Pathway Suppressor 2 (GPS2) Interacts with the Regulatory Factor X4 Variant 3 (RFX4_v3) and Functions as a Transcriptional Co-activator

RFX4_v3 (regulatory factor X4 variant 3) is a brain-specific isoform of the transcription factor RFX4. Insertional mutagenesis in mice demonstrates that Rfx4_v3 is crucial for normal brain development. Many genes involved in critical processes during brain morphogenesis are dysregulated in Rfx4_v3 mutant brains. For example, Cx3cl1 is a CX3C-type chemokine that is abundant in brain and is a direct transcriptional target of RFX4_v3 through a specific promoter X-box (X-box 1), the responsive element for RFX proteins. To identify potential interacting partners for RFX4_v3, we performed yeast two-hybrid analysis. Nine candidate interactors were identified, including GPS2 (G-protein pathway suppressor 2). Indirect immunofluorescence demonstrated that GPS2 and RFX4_v3 co-localized to the nucleus. Both GPS2 and RFX4_v3 mRNAs were also present in most portions of the adult mouse brain as well as in brains at different ages, suggesting that the two proteins could bind to each other. Co-immunoprecipitation assays indicated that physical interactions between GPS2 and RFX4_v3 did indeed occur. Furthermore, GPS2 was recruited to the Cx3cl1 promoter by RFX4_v3 and potentiated RFX4_v3 transactivation on this promoter through X-box 1, suggesting that the protein-protein interaction was functionally relevant. GPS2 bound to both the carboxyl-terminal region (amino acids 575-735) and the middle region (amino acids 250-574) of the RFX4_v3 protein. RFX4_v3 amino acids 1-574 stimulated the Cx3cl1 promoter to a similar extent as the full-length RFX4_v3 protein; however, deletion of the carboxyl-terminal region of RFX4_v3 impaired the co-activating abilities of GPS2. Based on these data, we conclude that GPS2 interacts with RFX4_v3 to modulate transactivation of genes involved in brain morphogenesis, including Cx3Cl1.

Induction of Cell Membrane Protrusions by the N-terminal Glutaredoxin Domain of a Rare Splice Variant of Human Thioredoxin Reductase 1

The human thioredoxin system has a wide range of functions in cells including regulation of cell proliferation and differentiation, immune system modulation, antioxidant defense, redox control of transcription factor activity, and promotion of cancer development. A key component of this enzymatic system is the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene. Transcription of TXNRD1 involves alternative splicing, leading to a number of transcripts also encoding isoforms of TrxR1 that differ from each other at their N-terminal domains. Here we have studied the TXNRD1_v3 isoform containing an atypical N-terminal glutaredoxin (Grx) domain. Expression of the transcript of this isoform was found predominantly in testis but was also detected in ovary, spleen, heart, liver, kidney, and pancreas. By immunohistochemical analysis in human testis with antibodies specific for the Grx domain of TXNRD1_v3, the protein was found to be predominantly expressed in the Leydig cells. Expression of the TXNRD1_v3 transcript was also found in several cancer cell lines (HCC1937, H23, A549, U1810, or H157), and in HeLa cells, it was induced by estradiol or testosterone treatments. Surprisingly, green fluorescent protein fusions with the complete TXNRD1_v3 protein or with only its Grx domain localized to distinct cellular sites in proximity to actin, and furthermore, had a potent capacity to rapidly induce cell membrane protrusions. Analyses of these structures suggested that the Grx domain of TXNRD1_v3 localizes first in the emerging protrusion and is then followed into the protrusions by actin and subsequently by tubulin. The results presented thus reveal that TXNRD1_v3 has a unique and distinct expression pattern in human cells and suggest that the protein can guide actin polymerization in relation to cell membrane restructuring.

Site-Specific Amino Acid Frequency, Fitness and the Mutational Landscape Model of Adaptation in Human Immunodeficiency Virus Type 1

Analysis of the intensely studied HIV-1 gp120 V3 protein region reveals that the among-population mean site-specific frequency of an amino acid is a measure of its relative marginal fitness. This surprising result may arise if populations are displaced from mutation-selection equilibrium by fluctuating selection and if the probability of fixation of a beneficial amino acid is proportional to its selection coefficient.

Identification of potential target genes for RFX4_v3, a transcription factor critical for brain development

Regulatory factor X4 variant transcript 3 (Rfx4_v3) gene disruption in mice demonstrated that interruption of a single allele (heterozygous, +/-) prevented formation of the subcommissural organ, resulting in congenital hydrocephalus, while interruption of two alleles (homozygous, -/-) caused fatal failure of dorsal midline brain structure formation. To identify potential target genes for RFX4_v3, we used microarray analysis to identify differentially expressed genes in Rfx4_v3-deficient mouse brains at embryonic day 10.5, before gross structural changes were apparent. Of 109 differentially expressed transcripts, 24 were chosen for validation and 22 were confirmed by real-time PCR. Many validated genes encoded critical proteins involved in brain morphogenesis, such as the signaling components in the Wnt, bone morphogenetic protein (BMP) and retinoic acid (RA) pathways. Cx3cl1, a CX3C-type chemokine gene that is highly expressed in brain, was down-regulated in the Rfx4_v3-null mice. Both human and mouse Cx3cl1 proximal promoters contained highly conserved X-boxes, known cis-acting elements for RFX protein binding. Using the Cx3cl1 promoter as an example of a target gene, we demonstrated direct binding of RFX4_v3 to the Cx3cl1 promoter, and trans-acting activity of RFX4_v3 protein to stimulate gene expression. These data suggest that RFX4_v3 may act upstream of critical signaling pathways in the process of brain development.

Expression of CD44 standard form and variant isoforms in non-small cell lung carcinomas

CD44, a cell adhesion molecule, has been implicated in tumor invasion and metastasis in certain malignancies. We studied the expression of CD44 standard (CD44s) and variant isoforms (CD44v) in 98 non-small cell lung carcinomas (NSCLCs) by immunohistochemistry and correlated the observations with clinical outcome. Formalin-fixed, paraffin-embedded archival tissues from 49 squamous cell carcinomas (SCCs) and 49 adenocarcinomas (ACs) were immunostained after microwave irradiation with monoclonal antibodies against CD44s and CD44v3, v4 5 , v6, v7 8 , and v10, and the results were correlated with histological tumor type, tumor stage, recurrence, and survival rates. SCCs of the lung showed strong membranous expression of each of the CD44s, v3, v4 5 , v6, and v10 proteins in comparison with ACs ( P < .0001). Staining for CD44 v4 5 was overwhelmingly positive in SCCs (72%) as compared with ACs (2.2%). Intense immunoreactivity for CD44v6 was present in 19 of 20 (95%) metastatic lung carcinomas. The bronchiolar basal cells and alveolar pneumocytes were positive for CD44s, v3, and v6. CD44s and variant isoform expression did not correlate with tumor stage, recurrence, and survival rates. In conclusion, there is significant immunopositivity of CD44s and variant isoforms in SCCs over ACs of the lung. Expression of CD44v6 may suggest an increased risk for local lymph node metastasis in NSCLCs. CD44v4/5 reactivity may be useful to discriminate squamoid differentiation in poorly differentiated NSCLCs.

Decreased levels of CD44 protein and mRNA in prostate carcinoma. Correlation with tumor grade and ploidy.

CD44, a transmembrane protein, is associated with cell-cell and cell-matrix interaction and with tumor growth and metastasis. Expression of both standard form and variant isoforms of CD44 protein has been associated with aggressive behavior and metastasis in various tumors, but has not been characterized in prostate adenocarcinoma (PAC). The expression of CD44 standard (CD44s) and splice variant v3, v4/5, v6, v7/8, and v10 proteins were studied in 109 PACs and correlated with tumor grade, DNA ploidy, and mRNA levels. Monoclonal antibodies against the various CD44 proteins were applied to microwave irradiated, formalin fixed, paraffin embedded sections. The DNA content of the tumors was evaluated by the Feulgen method with the CAS200 Image Analyzer. Total RNA exhibiting 18s and 28s bands was derived from two benign prostatic tissues and 5 PACs exhibiting decreased levels of CD44 protein by immunohistochemistry. The RNA was analyzed with reverse transcriptase polymerase chain reaction using CD44 specific primers. The basal cells of the benign prostatic acini revealed uniform membranous staining for CD44s, v3, and v6 in 95-97% of cases. Similar staining was observed for v4/5, v7/8, and v10 in 40%, 30%, and 2% of cases, respectively. Secretory epithelial cells of the benign prostatic acini showed predominant expression of CD44s (97% of cases). Staining for CD44 variant proteins (v3, v4/5, v6, v7/8, and v10) in this location ranged from 9-22% of cases. Approximately 70% of the PACs showed significant loss of CD44s expression, which correlated with high tumor grade (Gleason > or = 7) (P = 0.01) and aneuploid status (P = 0.002). In 93-98% of the PACS, there was a complete lack of membranous expression for all CD44 variant isoforms. The metastatic PACS did not show preferential expression of either the standard form or any variant isoform. The cDNA from the normal prostates yielded a prominent CD44 standard form polymerase chain reaction product at 482 base pair (bp) and variant isoforms at approximately 650 and 850 bp. No CD44 products could be amplified from the subset of five PAC cDNAs, even when present at four-fold excess. PACS exhibit down-regulation of CD44 protein expression, which correlates with high tumor grade and aneuploidy. v6 and v3 isoforms were preferentially expressed in the basal cells of benign prostatic acini. Based on a subset of cases, loss of CD44 protein expression is associated with decreased abundance of CD44 mRNA.

The temporal pattern of vitellogenin synthesis in Drosophila grimshawi

The temporal pattern of protein production and, in particular, vitellogenin protein synthesis during the sexual maturation of Drosophila grimshawi females has been studied in vivo by briefly feeding the flies with 35S-methionine and 3H-amino acids. The overall level of incorporation was very low in young flies; it then progressively increased to reach a maximum with the onset of sexual maturity at 13-15 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed three classes of proteins: those synthesized throughout the age spectrum, which constitute the majority of protein species; proteins synthesized primarily or only in young flies; and proteins synthesized only by the older flies. In this Drosophila species, the three vitellogenins (V1, V2, and V3) appeared to be synthesized in a two-phase pattern. In the first phase, small quantities of V1 and V2 were detected immunologically in the fat body and hemolymph of newly emerged and 1 day-old flies. These proteins did not accumulate in the hemolymph or the ovaries, apparently being unstable proteins. The second phase commenced in early vitellogenesis (7-9 days of age) with synthesis in the fat body of small quantities of V1 and V2, followed by V3 proteins. These proteins were secreted and accumulated in the hemolymph and 24 h later were found in the ovaries. Their quantities increased rapidly and a steady state of synthesis, release into the hemolymph, and uptake by the ovaries was reached by days 13-15. We have estimated that during the steady state of vitellogenin synthesis, a fly can synthesize in 24 h at least 152 micrograms of vitellogenins, which is more than 2% of its body weight, at an average rate of about 6.3 micrograms vitellogenins/h. About 2 micrograms of this are synthesized in the fat body, and about 4 micrograms in the ovaries. These findings are discussed in terms of their physiological implications and contrasted with the available data on Drosophila melanogaster.