ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ~130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.     

Human Papillomavirus Oncoproteins E6 and E7 Independently Abrogate the Mitotic Spindle Checkpoint

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G₁/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.     

A Reexamination of the Phylogenetic Position of Callimico (Primates) Incorporating New Mitochondrial DNA Sequence Data

The New World monkeys are divided into two main groups, Callitrichidae and Cebidae. Callimico goeldii shares traits with both the Cebidae and the Callitrichidae. Recent morphological phyletic studies generally place Callimico as the most basal member of the Callitrichidae. In contrast, genetic studies (immunological, restriction fragment, and sequence data) have consistently placed Callimico somewhere within the Callitrichidae, not basal to this clade. A DNA sequence data set from the terminal 236 codons of the mitochondrial ND4 gene and the tRNA^His, tRNA^Ser, and tRNA^Leu genes was generated to clarify the position of Callimico. The sequences of 887 base pairs were analyzed by maximum-parsimony, neighbor-joining, and maximum-likelihood methods. The results of these various methods are generally congruent and place Callimico within the Callitrichidae between the marmosets (Callithrix and Cebuella) and the tamarins (Saguinus and Leontopithecus). Combined analyses of all suitable nuclear and mitochondrial gene sequences confirm the position of Callimico between the marmosets and the tamarins. As available molecular evidence indicates that Callimico is more closely related to the marmosets than to the tamarins, a reconsideration of the morphological evidence in light of the consensus tree from DNA sequence analyses is warranted. The marmosets and tamarins share four morphological characters (loss of the third molar, loss of the hypocone, reduced body size, reproductive twinning). Dwarfism may have evolved repeatedly among the Callitrichidae. It is well-known that the loss of a character can occur many times independently. The reproduction of marmosets and tamarins is extremely specialized and it is difficult to imagine that this complex and unique twinning system evolved separately in marmosets and tamarins. However, it is possible that a secondary reversal to single offspring took place in Callimico. Received: 20 March 1997 / Accepted: 17 December 1997     

Evidence that humidity influences snake activity patterns: a field study of the Malayan pit viper Calloselasma rhodostoma

Multivariate statistical methods were used to elucidate which environmental factors influence the activity patterns of free-living Malayan pit vipers, Calloselasma rhodostoma. Fourteen adult snakes were implanted with miniature radiotransmitters and located a total of 887 times in 5 months. The pit vipers usually remained coiled on the ground for several consecutive days before moving at night to a new site. Partial correlation tests revealed that the frequency and distance of movements to new sites by tagged snakes were highly positively correlated with ambient relative humidity, but not with rainfall, ambient temperature or the lunar cycle. This finding was corroborated by the frequency with which active non-tagged C. rhodostoma were encountered at night, In each site, the proportion of the snakes' bodies exposed to view was positively correlated with ambient humidity, and the snakes retreated to areas with deeper undergrowth when ambient humidity was low. Overt thermoregulatory behaviour was not observed, and implanted thermosensitive transmitters revealed that the snakes were passive thermoconformers.
These findings seem lo contradict much of the current literature which shows temperature to be the dominant abiotic factor affecting reptilian activity, but most herpetologists have considered only temperate forms. Ambient temperature in our tropical study site was warm and relatively constant throughout the year (mean daily range = 24- 33°C), so the pit vipers could passively maintain body temperature within a fairly narrow range, with a daytime mean of 29.4°C. Ambient relative humidity, on the other hand, was very variable, and confining exposure and activity to periods of high ambient humidity may be necessary to avoid dehydration     

Branch growth of riparian cottonwoods: a hydrologically sensitive dendrochronological tool

 The conservation of riparian (river valley flood plain) forests relies on the provision of instream flows that are sufficient to sustain tree growth. In the present study, annual branch growth increments were investigated as an indicator of environmental favorability for riparian cottonwoods. Trees of three species, Populus angustifolia, P. balsamifera, and P. deltoides, and their natural interspecific hybrids, were studied at five sites along the Oldman and South Saskatchewan rivers in Alberta, Canada. Annual branch growth increments for the interval from 1983 to 1992 were positively correlated with stream flows (r ² = 0.79 at Lethbridge) and slightly negatively correlated with weather variables that contribute to water demand: evaporation, temperature, wind, and/or sunshine. The combination of January to May stream flow (water supply) and June evaporation (water demand) almost entirely accounted for the branch growth variation across years (r ² = 0.91 at Lethbridge). Tree ring increments were also investigated but were less closely correlated than branch increments across trees or with stream flow. Branch growth increments thus provide an accurate but short duration (1 or 2 decades) record of environmental favorability for growth. The close correlation between branch growth and stream flow indicates that water is the principal limitation to growth of these riparian cottonwoods and that these trees obtained their water from a source linked to the stream, the riparian water table. Analyses of branch increments should provide a management tool for (i) determining instream flow needs for riparian cottonwoods and (ii) analyzing impacts of stream flow alterations due to river damming or water diversion. Received: 8 May 1997 / Accepted: 23 September 1997     

Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis

The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of α1 → 2, α1 → 3, α1 → 3 linked D -rhamnose (Rha). The A-band polysaccharide is assembled by the α-D -rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two α1 → 3 linked Rha residues and one α1 → 2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3′ end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3′ end of the cluster and are predicted to encode a co-enzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D -galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.     

Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium

The mouse intestinal epithelium undergoes rapid renewal throughout life, thereby requiring continuous coordination of its cellular proliferation, differentiation, and death programs. Recent advances in our understanding of this process have highlighted some of the molecules that regulate renewal and their potential roles in gut neoplasia.