Cytomegalovirus infection via mother's milk: could distinct virus strains determine different disease patterns in preterm twins?

We describe a case of cytomegalovirus infection via the mother's milk in preterm twins who showed a different disease pattern. Molecular analyses to genotype the viral genome disclosed a mixed population of virus strains in the milk. These isolates were differentially transmitted to each of the twins and this may be responsible of the different patterns of disease.     

p16(INK4A) immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus.

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16(INK4A) tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16(INK4A) suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16(INK4A) expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA-ISH), gave high-intensity p16(INK4A) immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.     

Morphology and genetics of a Babesia isolate from Capreolus capreolus

A Babesia isolate that was morphologically distinct from Babesia capreoli and very similar to B. divergens was found in the blood of a roe deer (Capreolus capreolus) found dead in central Italy. Sequences corresponding to the full coding region of the 18S ribosomal RNA (rRNA) gene were identical to a sequence reported for Babesia divergens from a reindeer (Rangifer tarandus) and 99.9% and 99.8% similar to those reported for B. capreoli and bovine origin B. divergens, respectively.     

Lectin histochemistry for in situ profiling of rat colon sialoglycoconjugates

The growing interest in glycoconjugates expressed and released by the epithelium of the intestinal mucosa is tightly related to the multiple functional roles attributed to sialic acid and its derivatives. In the present work, biotin and HRP conjugated lectins were used to detect the sialylation pattern and to identify specific structural features of sialoderivatives in the rat colon. In particular, the occurrence and distribution of sialic acids linked alpha2,6 to D-Gal/D-GalNAc and alpha2,3 to D-Gal were directly demonstrated with SNA and MAL II binding, respectively. In addition, in order to by-pass the specificity problems of SNA and MAL II as histochemical reagents, as well as to look for additional and complementary information about acetylation degree and sites, we combined sialidase digestion, potassium hydroxide deacetylation, and differential periodate oxidation with PNA and DBA binding. The data showed the distribution and structure of sialic acid-beta-D-Gal(1-3)-D-GalNAc and sialic acid-D-GalNac sequences, which proved to be widely distributed as cellular components or secretory products in surface goblet cells and crypt cells of the colonic epithelium. A high degree of O-acetylation, with acetyl groups mainly at 9 and 4 positions, was found, showing an increasing gradient from the proximal to distal portion of the colon. These results, which largely reproduce the sialylation pattern in other species, contribute new insights in defining the tissue specific expression of sialoderivatives in the colonic mucosa, and testify to their high heterogeneity which the wide range of sialic acid functional correlates in the intestinal tract depend on.     

Mechanism of mitosis-specific activation of MEK1

Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well established roles in entry and exit from mitosis in Xenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G(2)/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis.     

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.     

Centrosomal and cytoplasmic Cdc2/cyclin B1 activation precedes nuclear mitotic events

The activation of cdc2/cyclin B is the trigger for entry into mitosis. The mechanism of cdc2/cyclin B activation is complex, but the final step is the dephosphorylation of the Thr14 and Tyr15 residues on the cdc2 subunit, catalyzed by a member of the Cdc25 family of phosphatases. Cdc2/cyclin B1 accumulates at the centrosome in late G2 phase and has been implicated in the conversion of the centrosome from an interphase to a mitotic microtubule organizing center. Here we demonstrate biochemically that cdc2/cyclin B1 accumulates at the centrosome in late G2 as the inactive, phosphotyrosine 15 form and that the centrosomal cdc2/cyclin B1 can be activated in vitro by recombinant cdc25B. We provide evidence that a portion of the cdc2/cyclin B1 translocated into the nucleus in prophase is the inactive tyrosine-15-phosphorylated form. At this time the centrosomal and cytoplasmic cdc2/cyclin B1 is already active. This provides evidence that the activation of cdc2/cyclin B1 is initiated in the cytoplasm and that full activation of the translocated pool occurs in the nucleus.     

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.     

Generation of a Genome Scale Lentiviral Vector Library for EF1α Promoter-Driven Expression of Human ORFs and Identification of Human Genes Affecting Viral Titer

The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.