Caenorhabditis elegans embryonic axial patterning requires two recently discovered posterior-group Hox genes.

Hox genes encode highly conserved transcription factors that control regional identities of cells and tissues along the developing anterior-posterior axis, probably in all bilaterian metazoans. However, in invertebrate embryos other than Drosophila, Hox gene functions remain largely unknown except by inference from sequence similarities and expression patterns. Recent genomic sequencing has shown that Caenorhabditis elegans has three Hox genes of the posterior paralog group [Ruvkun, G. & Hobert, O. (1998) Science 282, 2033-2041]. However, only one has been previously identified genetically, and it is not required for embryonic development [Chisholm, A. (1991) Development (Cambridge, U.K.) 111, 921-932]. Herein, we report identification of the remaining two posterior paralogs as the nob-1 gene and the neighboring php-3 gene. Elimination of nob-1 and php-3 functions causes gross embryonic defects in both posterior patterning and morphogenetic movements of the posterior hypodermis, as well as posterior-to-anterior cell fate transformations and lethality. The only other Hox gene essential for embryogenesis is the labial/Hox1 homolog ceh-13, required for more anterior patterning [Brunschwig, K., Wittmann, C., Schnabel, R., Burglin, T. R., Tobler, H. & Muller, F. (1999) Development (Cambridge, U.K.) 126, 1537-1546]. Therefore, essential embryonic patterning in C. elegans requires only Hox genes of the anterior and posterior paralog groups, raising interesting questions about evolution of the medial-group genes.

Modeling regulatory networks with weight matrices.

Systematic gene expression analyses provide comprehensive information about the transcriptional response to different environmental and developmental conditions. With enough gene expression data points, computational biologists may eventually generate predictive computer models of transcription regulation. Such models will require computational methodologies consistent with the behavior of known biological systems that remain tractable. We represent regulatory relationships between genes as linear coefficients or weights, with the "net" regulation influence on a gene's expression being the mathematical summation of the independent regulatory inputs. Test regulatory networks generated with this approach display stable and cyclically stable gene expression levels, consistent with known biological systems. We include variables to model the effect of environmental conditions on transcription regulation and observed various alterations in gene expression patterns in response to environmental input. Finally, we use a derivation of this model system to predict the regulatory network from simulated input/output data sets and find that it accurately predicts all components of the model, even with noisy expression data.

Novel gene expression mechanism in a fission yeast retroelement: Tf1 proteins are derived from a single primary translation product.

In sharp contrast to the single ORF of the Schizosaccharomyces pombe retrotransposon Tf1, retroviruses and most retrotransposons employ two different ORFs to separately encode the Gag and Pol proteins. The different ORFs are thought to allow for overexpression of the Gag protein relative to Pol protein presumed necessary for the assembly of functional retrovirus particles and virus-like particles (VLPs). The results of in vivo experiments designed to detect the transposition of Tf1 show that Tf1 is indeed active and can insert itself into the host genome via a true retrotransposition process. Thus, a paradox emerged between the lack of any obvious means of overexpressing Tf1 Gag protein and the demonstrated functionality of the element. Epitope tagging experiments described here confirm that the Tf1 large ORF is intact and that there is no translational or transcriptional mechanism used to overexpress the Tf1 Gag protein. In addition, we used sucrose gradients and antisera specific for Tf1 capsid (CA) and integrase (IN) to show that the Tf1 proteins do assemble into uniform populations of macromolecular particles that also cosediment with Tf1 reverse transcription products. This evidence suggests that Tf1 proteins form VLPs without using the previously described mechanisms that retroviruses and retrotransposons require to overexpress Gag proteins.

Sequence analysis of closely related retrotransposon families from fission yeast.

Two families of retrotransposons, Tf1 and Tf2, have been isolated from the fission yeast, Schizosaccharomyces pombe. We report here the nucleotide (nt) sequence of a Tf2 element, the only retrotransposon family known from the commonly used laboratory strains, 972 and 975, and their derivatives. The total nt sequence of Tf2 was derived from the complete sequence of the coding region and 3' long terminal repeat (LTR) of randomly cloned element Tf2-1, and from a full 5' LTR and approximately one-third of the open reading frame (ORF) of Tf2-43, a Tf2 element found in the head-to-head orientation adjacent to the Sz. pombe rpb6 gene. The two Tf2 sequences are nearly identical and both of them contain a single ORF encoding a protein with regions of sequence similar to protease, reverse transcriptase, RNase H (RH) and integrase from other retrotransposons and retroviruses. Sequence comparisons between Tf1 and Tf2 indicate an extreme divergence of the putative capsid protein-encoding regions of these two elements, as well as divergence of a segment of the LTR, but otherwise virtually identical sequence.

Evaluation of an outbreak of foodborne illness initiated in the emergency department.

STUDY OBJECTIVES: For many foodborne outbreaks, the pathogen and food vehicle never are identified. Delayed collection of epidemiologic and microbiologic information may contribute to this. We postulated that collection of this information from ill persons as they presented to the emergency department during a recent outbreak might contribute to earlier identification of the pathogen and vehicle. DESIGN: At least 690 of 1,900 conventioneers developed gastrointestinal symptoms after attending a banquet. A questionnaire was developed to collect information on specific food histories, incubation periods, symptoms, physical findings, and demographics. These results were compared with results of investigations by the city and state departments of public health. SETTING: The ED of Rush-Presbyterian-St Luke's Medical Center, a tertiary care university hospital in Chicago, Illinois. TYPE OF PARTICIPANTS: Adults (24 men and nine women) presenting to the ED with gastrointestinal symptoms after eating a common meal. MEASUREMENTS AND MAIN RESULTS: The clinical syndrome suggested an invasive pathogen. Based on this, clinical microbiology laboratory procedures were modified (isolation plates were reviewed during the evening shift). This led to early identification of the first isolates (Salmonella enteritidis) from the outbreak. The questionnaire also narrowed the vehicle to one of two foods served. Investigations by the departments of public health subsequently identified one of these, bread pudding with a raw egg based-sauce, as the vehicle. CONCLUSION: Outbreak evaluations can begin in the ED or any other patient care facility. This evaluation need not always add significantly to the expenditure of time, manpower, or laboratory studies. The evaluation of even a small percentage of ill persons from a large outbreak may provide useful epidemiologic information and be particularly important in settings with limited public health resources.

Candida krusei fungemia. Report of 4 cases and review of the literature.

Candida krusei has recently been increasingly recognized as a pathogen in immunocompromised patients with malignancies. We report four immunocompromised patients with C. krusei fungemia and review the literature. Including our 4 cases, 62 cases of C. krusei fungemia were identified in the literature. Detailed information was available for only 25 patients. The clinical features of patients with C. krusei fungemia are similar to those reported for Candida tropicalis. Most patients were neutropenic and more than one half of the patients had received antifungal therapy and had evidence of gastrointestinal mucosal breakdown before the development of C. krusei fungemia. The overall mortality was 48%. Patients treated with regimens containing amphotericin B had improved survival over patients who received no therapy. Favorable response rates were higher in patients receiving high-dose amphotericin B or high-dose amphotericin B plus flucytosine when compared to patients treated with low-dose amphotericin B.

Transient porphyrinemia in a liver transplant recipient.

We report a case of acquired porphyrinemia in a liver transplant recipient. Thrombosis of the portal vein and hepatic artery led to massive necrosis of the patient's first donor liver, necessitating its removal. After an 18-hour anhepatic period, a second donor liver was transplanted. During this interval, the patient was warmed with an infrared heat lamp that emitted bright visible light. Following the anhepatic period, a photodistributed cutaneous erythema was noted. The level of total serum porphyrins during the anhepatic period was significantly elevated at 1.548 mumol/L (normal, less than 0.018 mumol/L). Fractionation showed that the levels of the hydrophobic porphyrins, coproporphyrin and protoporphyrin, were elevated, while the levels of the hydrophilic porphyrins remained normal. Following the successful second liver transplantation, the patient's porphyrin levels declined. This is the first reported case of acquired porphyrinemia in a liver transplant recipient.

Two related families of retrotransposons from Schizosaccharomyces pombe.

Two related families of transposons were isolated from schizosaccharomyces pombe, an organism which has been the object of extensive genetic studies which had previously produced no evidence for the existence of such elements. These two classes of repeated DNAs, dubbed Tf1 (transposon of fission yeast 1) and Tf2 have many properties of retrotransposons. Tf1 and Tf2 both possess long terminal repeats and predicted protein sequences that resemble the protease, reverse transcriptase, and integrase domains of retroviruses. The chromosomal locations and total numbers of Tf1 and Tf2 differ greatly in various isolates of S. pombe. The Tf elements are expressed in the form of 4.5-kb mRNAs. The complete sequence of Tf1 was determined and suggests that a novel mechanism for regulating its gene expression may be used.

Site specificity in vimentin-membrane interactions: intermediate filament subunits associate with the plasma membrane via their head domains.

Fragments of vimentin, generated by chemical or enzymatic cleavages, were analyzed for their capacity to bind to human inverted erythrocyte membrane vesicles. Only peptides comprising the amino-terminal head domain of vimentin molecules were competent in associating with the membranes. In vitro studies also demonstrated that isolated ankyrin (the major vimentin acceptor site on the membrane) binds to an oligomeric species of vimentin and prevents the formation of characteristic 10-nm filaments. These data, taken together with the observation that the NH2-terminal end of vimentin is implicated in the polymerization process (Traub, P., and C. Vorgias, J. Cell Sci., 1983, 63:43-67), imply that intermediate filaments may contact the membrane in an end-on fashion, using the exposed head domains of their terminal subunits.

The structure of a small collagenous fragment isolated from chicken hyaline cartilage.

In previous experiments, two collagenous fragments were isolated from pepsin digests of chicken hyaline cartilage and called the high molecular weight, (HMW) and low molecular weight (LMW) fractions [3]. In the present experiments, the chains of LMW were isolated after denaturation and subsequent reduction and alkylation of interchain disulfide bridges and were further fractionated by carboxymethyl-cellulose chromatography. Four peaks were resolved during chromatography and were designated LMW 1, 2A, 2B, and 3. Amino acid analyses and peptide mapping after cleavage with trypsin, V8 protease, and cyanogen bromide showed that three genetically distinct chains must be present in LMW. Fractions 2A and 2B were very similar, but not identical, in structure. LMW 1, 2A plus 2B, and 3 were consistently isolated in approximately equal proportions, suggesting that the probable chain organization of LMW is [1][2A + 2B][3]. This suggestion was supported further by experiments that attempted to fractionate LMW by carboxymethyl-cellulose chromatography after denaturation but without reduction and alkylation of interchain disulfide bridges. No fractionation of LMW was achieved, the single peak subsequently being shown to contain LMW 1, 2A plus 2B, and 3.

The structural basis of ankyrin function. I. Identification of two structural domains.

The structure of ankyrin, a major linking protein between spectrin and the erythrocyte membrane, was analyzed after restricted proteolytic digestion at 0 degree C. By the use of two-dimensional peptide mapping, we found that tryptic digestion of ankyrin (1 h, 0 degree C) resulted in the production of two nonoverlapping peptides of molecular weights 82,000 and 55,000. The 82,000-dalton peptide had a basic isoelectric point (7.9) and was remarkably sensitive to further proteolytic digestion; after 24 h at 0 degree C, trypsin completely digested this peptide into fragments too small to detect by gel electrophoresis. The 55,000-dalton peptide was neutral (isoelectric point = 6.9-7.2) and more resistant to further proteolytic cleavage. After a 24-h digestion with trypsin at 0 degrees C, the 55,000-dalton peptide was cleaved into two complementary fragments of molecular weight 32,000 and 15,000. Analysis of phosphorylated ankyrin indicated that the phosphates were exclusively found in these two complementary peptides. By comparison with larger fragments, we were able to align the constituent peptides of ankyrin and propose a low resolution model. Ankyrin appears to be a bipolar molecule containing a basic domain of 82,000 daltons and a neutral phosphorylated domain of 55,000 daltons.

The structural basis of ankyrin function. II. Identification of two functional domains.

Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.

Alloxan stimulation and inhibition of insulin release from isolated rat islets of Langerhans.

The rate of alloxan-induced insulin release was measured from rat islets maintained in a simple perifusion system. Insulin release during the five-minute exposure to alloxan reached its maximum rate after two to three minutes of the exposure and then rapidly declined. This insulin release was dependent upon extracellular calcium and was associated with an increased 45Ca uptake by isolated islets. Once exposed to alloxan, however, the islets did not release insulin when stimulated again with D-glucose or alloxan. These effects of alloxan on insulin release (stimulation and subsequent inhibition) and the increased 45Ca uptake were prevented by the presence of 3-0-methyl-D-glucose during the alloxan exposure. These findings indicate a close correlation between alloxan-induced insulin release and the subsequent inhibition of further insulin release. D-glucose, when present during the entire five-minute exposure to alloxan, protected competitively against alloxan inhibition of insulin release. In addition, D-glucose, when present immediately after brief (one to three minutes) alloxan exposures, reversed some of the subsequent inhibition of insulin release. These findings suggest that alloxan and D-glucose were competing for a common site on the beta-cell. The possibility of this site being a receptor responsible for the initiation of insulin release is discussed.

Alloxan uptake by isolated rat islets of Langerhans.

Alloxan inhibits subsequent glucose-induced insulin release from isolated rat islets of Langerhans maintained in vitro. Several agents (D-glucose, D-mannose, 3-0-methyl-D-glucose, caffeine, and cytochalasin B) when present during the alloxan exposure protect against alloxan inhibition of insulin release. To examine the mechanism of alloxan inhibition, the uptake of [2-14C]alloxan was measured in isolated islets. [2-14C]Alloxan was rapidly accumulated by the islets in a time- and temperature-dependent manner. The radio-activity from islets incubated with [2-14C]alloxan was isolated and shown by thin layer chromatography to comigrate with alloxan and alloxanic acid, an alloxan decomposition product. As no uptake of radioactivity occurred in the presence of medium containing the radioactive decomposition product, it was concluded that alloxan enters the intracellular space of the islet and undergoes a subsequent internal decomposition. Some of the protective agents (3-0-methyl-D-glucose, caffeine, and cytochalasin B) partially inhibited alloxan uptake, whereas others (D-glucose and D-mannose) increased the uptake of alloxan. These and other results suggest that the experimental agents do not provide protection against alloxan inhibition by preventing the entry of alloxan into the intracellular space of the islet. The possibility of D-glucose and alloxan competing for a common binding site on the cell membrane is discussed.

Mechanism of barbituric-acid protection against inhibition by alloxan of glucose-induced insulin release.

Isolated rat islets were maintained in a simple static incubation system and were exposed to alloxan for a period of five minutes. Alloxan inhibited subsequent glucose-induced insulin release in a dose-dependent manner at 37 degrees C., with 650 muM alloxan producing 94 per cent inhibition of insulin release. Barbituric acid, a compound structurally related to alloxan, provided complete protection (at 37 degrees C.) against this inhibition of insulin release when present during the alloxan exposure. At 23 degrees C., barbituric acid was shown to be absent from the intracellular space of the islet yet still protected completely against alloxan inhibition of insulin release. Thus, barbituric acid apparently provided protection against alloxan in the extracellular medium. By fluorometric and chromatographic analyses, it was determined that barbituric acid reacted rapidly with alloxan to produce a new compound. These findings indicate that barbituric acid protected against alloxan by a chemical reaction in the medium.