New ways to study developmental genes in spore-forming bacteria.

The regulated activation of numerous sets of genes in multiple chromosomal locations is a hallmark of cellular differentiation in both eukaryotes and prokaryotes. Certain species of bacteria that experience complex developmental cycles are especially attractive as systems in which to study the mechanisms of this kind of gene regulation because they are highly amenable to both biochemical and genetic approaches. Bacillus subtilis, which undergoes extensive cellular differentiation when it sporulates, is one such system. Many new methods are now available in this Gram-positive species for identifying, manipulating, and studying the regulation of genes involved in spore formation, including the use of transposable genetic elements that create gene fusions in vivo as an automatic consequence of insertions into genes.

DNA binding domain and subunit interactions of transcription factor IIIC revealed by dissection with poliovirus 3C protease.

Transcription factor IIIC (TFIIIC) is a general RNA polymerase III transcription factor that binds the B-box internal promotor element of tRNA genes and the complex of TFIIIA with a 5S rRNA gene. TFIIIC then directs the binding of TFIIIB to DNA upstream of the transcription start site. TFIIIB in turn directs RNA polymerase III binding and initiation. Human TFIIIC contains five different subunits. The 243-kDa alpha subunit can be specifically cross-linked to B-box DNA, but its sequence does not reveal a known DNA binding domain. During poliovirus infection, TFIIIC is cleaved and inactivated by the poliovirus-encoded 3C protease (3Cpro). Here we analyzed the cleavage of TFIIIC subunits by 3Cpro in vitro and during poliovirus infection of HeLa cells. Analyses of the DNA binding activities of the resulting subcomplexes indicated that an N-terminal 83-kDa domain of the alpha subunit associates with the beta subunit to generate the TFIIIC DNA binding domain. Cleavage with 3Cpro also generated an approximately 125-kDa C-terminal fragment of the alpha subunit which remained associated with the gamma and epsilon subunits.

Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis.

The ctc gene of Bacillus subtilis is transcribed in vitro by the minor RNA polymerase holoenzyme forms, E sigma 37 and E sigma 32. To study the expression and regulation of ctc in vivo, we constructed operon and translational fusions of the ctc promoter region to the lacZ gene of Escherichia coli. Our results indicate that ctc is regulated at the transcriptional level, and that this RNA synthesis is maximally induced at the end of the exponential phase of growth under nutritional conditions which inhibit the activity of the tricarboxylic acid cycle. Analysis of in vitro-constructed deletion mutations extending into the ctc promoter region demonstrated that the region required for this regulation is no greater than 53 base-pairs in length. We also compared the expression of ctc to that of another B. subtilis gene, which is transcribed by E sigma 37 and E sigma 32 in vitro, the sporulation gene spoVG. Although the ctc and spoVG promoter regions are recognized by the same forms of RNA polymerase in vitro, our results show that they differ strikingly in the nutritional and genetic requirements for their expression in vivo.

Identification of the bases in the ompF regulatory region, which interact with the transcription factor OmpR.

Expression of the outer membrane protein OmpF of Escherichia coli K-12 is influenced by a variety of environmental signals. Most of the signals are thought to regulate OmpF expression at the level of transcription initiation. A key element of this regulation is the interaction between the transcriptional factor OmpR and the cis-acting regulatory region of ompF. In this study, we used a combination of DNase I, dimethyl sulfate and hydroxyl radical footprinting analysis and DNA migration retardation assays to identify the bases within the ompF regulatory region that are in contact with OmpR. Our results indicate that the -107 to -39 region of ompF contains three individual binding sites and that a single OmpR-binding site is capable of interacting with two OmpR molecules. We also establish that a single OmpR-binding site is composed of two half-sites and that both half-sites are required for the formation of stable OmpR/DNA complexes. Comparisons of the sequences protected by OmpR indicate that an OmpR-binding site spans approximately 18 bp and has two highly conserved G/C base-pairs that are separated by three nucleotides. Although the three OmpR-binding sites we identified exhibit limited sequence similarity, this may reflect the fact that two of the sites are incapable of binding OmpR independently and can bind OmpR only if adjacent to another OmpR-binding site. Finally, our DNA migration retardation assays suggest that phosphorylation stimulates the cooperative interactions between OmpR molecules bound at neighboring sites. Therefore, this study provides a detailed understanding of how OmpR interacts with its binding sites immediately upstream of ompF and serves as a foundation for studying how phosphorylation of OmpR results in the regulation of ompF expression in response to environmental signals.

Tenderness assessments of top loin steaks from retail markets in four U.S. cities.

The purpose of this study was to evaluate the tenderness of beef loin steaks from retail markets in 4 U.S. cities. Beef top loin steaks ( = 1,613) were obtained for Warner-Bratzler shear force (WBSF), slice shear force (SSF), and consumer sensory determinations. Personnel at 4 universities (California Polytechnic State University, Colorado State University, University of Missouri, and Texas A&M University) conducted the study over a 12-mo period. Enhanced/blade-tenderized top loin steaks had the lowest ( < 0.05) WBSF and SSF values, whereas nonenhanced top loin, bone-in steaks had the highest ( < 0.05) WBSF and SSF values. Enhanced/blade-tenderized top loin steaks received the highest ( < 0.05) ratings by consumers for palatability scores, whereas nonenhanced top loin, bone-in steaks had the lowest ( < 0.05) consumer panelist ratings. The USDA quality grade did have an effect ( < 0.05) on the tenderness of nonenhanced steaks but did not affect ( > 0.05) steaks that were enhanced/blade tenderized. The WBSF values and consumer sensory values for top loin steaks were comparable to the 2010 National Beef Tenderness Survey, signifying that no drastic changes in tenderness have occurred due to changes in antemortem or postmortem conditions.

Hereditary non-progressive torsion dystonia with intellectual disturbance.

Three siblings of a consanguineous parents with involuntary movements are reported. The mother had only a very slight neck tremor, without any other neurological abnormality, and the father had died. The 38-year-old son (Case 1) complained of involuntary movements at the age of 6. His involuntary movements were observed in the tongue, perioral region and upper and lower extremities: jerky movements with dystonic features. The 46-year-old elder brother (Case 2) experienced involuntary movements at the age of 18. Involuntary movements were observed in the upper extremities; he also had torticollis and tremulous movements in the neck, and jerky movements in the perioral region. They showed gait disturbance and dysarthria. The 35-year-old sister (Case 3) also experienced involuntary movements. When she was writing, her involuntary movements were obvious: dystonia and myoclonic jerks. Tremor in the neck was also seen. Their intelligence was below average. We concluded that this family had hereditary torsion dystonia, with myoclonus, and low intelligence. This condition may be associated with an autosomal recessive gene.

[A case report of miliary tuberculosis with tubercular meningitis diagnosed and followed by polymerase chain reaction method].

We report here a case of miliary tuberculosis with tubercular meningitis in which the polymerase chain reaction (PCR) amplification method was useful for its rapid diagnosis and follow up. A 70 year old male was hospitalized for further examination and treatment of diffuse small nodular shadows on the chest X-ray. After receiving antimicrobial therapy shadows still remained and he gradually lost visual acuity. He had no meningeal signs, and no remarkable finding on cranial CT. Cerebrospinal fluid examination showed increased cell number with predominantly lymphocytes. Cranial MRI (Gd DTPA) showed lateral ventricular ependymitis. Pulmonary tuberculosis and secondary tubercular meningitis were suspected, but we failed to detect microorganisms from the cerebrospinal fluid, gastric juice, sputum, and urine by the conventional method. However, by the polymerase chain reaction amplification method specific DNA fragments of Mycobacterium tuberculosis complex was detected from the cerebrospinal fluid, gastric juice, bronchoalveolar lavage fluid and serum. The final diagnosis of miliary tuberculosis with tubercular meningitis was established. We administered antitubercular drugs and observed the clinical course. He recovered and the polymerase chain reaction showed negative consequences in all samples. The judgement of PCR and the clinical course were compatible and parallel with the clinical course.

[Serial observations of respiratory function in disabled patients with myotonic dystrophy].

Serial assessments of respiratory function were performed over 1 to 8 years in 12 patients with myotonic dystrophy. Respiratory parameters included vital capacity (percentage of predicted value; %VC) and one-second forced expiratory volume (percentage of predicted value; FEV1.0%) on spirogram and arterial blood gases at rest. The patients who had no primary respiratory disease were ranged in age from 31 to 61 years, with an average age of 47 years, and their mean duration of illness was 20 years. All the subjects were severely disabled; only two patients were able to walk with assistance, but other ten patients were confined to a wheelchair. During the observation period, five patients died; 4 due to respiratory failure and one cardiac and respiratory failure. The following results were obtained: (1) almost all the patients showed a reduction in %VC, the severity of which was gradually progressed with advancing age. A significant negative correlation was observed between %VC and duration of illness. (2) PaCO2 was negatively correlated with %VC, while the relationship between PaO2 and %VC appeared to be a significant positive correlation. Chronic alveolar hypoventilation (hypercapnia and hypoxemia) was particularly likely when vital capacity (VC) was less than 40% of the predicted value in the patients. This study indicates that a reduction of VC plays an important role in development of respiratory failure in myotonic dystrophy and serial measurements of VC and arterial blood gases are useful in the detection of it.

[Prevalence and founder effect of Huntington's disease in the San-in area of Japan].

After the DNA diagnosis, we evaluated the prevalence of Huntington's disease (HD) in the San-in area of Japan, and confirmed the founder effect. The population of the area was 1,387,000 on October 1st, 1993. There were 10 patients with HD in the San-in area, who were diagnosed clinically. They all had involuntary movement, mental disturbance, changes of character and atrophy of the caudate nucleus. However, one of the patients showed no positive family history of HD. The other nine patients had members with HD in their families, although those family members of the patients had already died. The expansion of the CAG repeat was observed in nine of the patients. In the patient who had no positive family history, expansion of the CAG repeat was not seen. According to the nine patients with expansion of the CAG repeat, the prevalence was 0.65/100,000. Haplotypes using polymorphism markers of D4S111 and D4 S136 were studied. The haplotypes which were observed in only 2.7% of the normal population were shown in all nine HD patients. Thus, the obvious disequilibrium was seen. These results suggested a common ancestor of these HD patients.

EnvZ, a transmembrane environmental sensor of Escherichia coli K-12, is phosphorylated in vitro.

By fusing the transcriptional and translational start signals of lacZ to envZ, we have obtained high-level synthesis of a truncated EnvZ protein (EnvZ115) in which the first 38 amino acids of EnvZ are replaced with the first 8 amino acids of LacZ. Using this construct, we have partially purified the EnvZ115 protein and demonstrated that this protein can be phosphorylated in vitro. We suggest that phosphorylation may be an important feature of EnvZ function.

Mutations in the alpha subunit of RNA polymerase that affect the regulation of porin gene transcription in Escherichia coli K-12.

The two-component regulatory system consisting of OmpR and EnvZ controls the differential expression of major outer membrane porin proteins OmpF and OmpC of Escherichia coli K-12. We have isolated and characterized two mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that decrease the expression of OmpF. These mutations have a number of properties that distinguish them from previously isolated rpoA mutations that affect porin expression. The rpoA203 mutation decreases the expression of porin genes ompF and ompC and also decreases the expression of the malE and phoA genes. In contrast, rpoA207 decreases the expression of ompF but does not affect ompC, malE, or phoA transcription. Our results suggest that mutations at various positions in the alpha subunit may affect the OmpR-dependent transcription of ompF and ompC differently and may be useful for analyzing the mechanism underlying their differential expression in response to medium osmolarity.

An epidemiologic study of cerebrovascular disease in western Japan: with special reference to transient ischemic attacks.

The prevalence and incidence ratios of cerebrovascular disease, with special reference to transient ischemic attack (TIA), were studied in the towns of Daisen and Ama in western Japan. There have been no previous reports on this subject in Japan. The prevalence ratios of TIA were estimated to be 4.4 in Daisen and 2.0 in Ama per 1,000 people over 40 years old. The ratio of carotid arterial system TIA to vertebrobasilar arterial system TIA was about 1 to 1. The incidence ratios of stroke were 319.6 in Daisen and 314.5 in Ama per 100,000 people of all ages. The prevalence ratios of stroke were estimated to be 14.8 in Daisen and 13.5 in Ama per 1,000 people of all ages. The prevalence ratio of TIA in Japan is about one-third to one-half of that in Western countries. However, the prevalence of complete stroke is much higher in Japan compared with that in Western countries. Therefore, the ratio of TIA to stroke is much lower in Japan than in Western countries. The obstruction of small intracranial arteries, in addition to heart disease, might play an important role in TIA in Japan, whereas in Western countries TIA might be mostly caused by heart disease or the atherosclerosis of extracranial arteries.

Differential expression of the OmpF and OmpC porin proteins in Escherichia coli K-12 depends upon the level of active OmpR.

We have generated a mutant form of the OmpR regulatory protein, OmpRD55E, that is active independent of the EnvZ kinase. Notably, the pattern of OmpF and OmpC expression can be altered simply by changing the level of this mutant protein in the cell. This result supports a key prediction of the current model of porin regulation, which states that the differential regulation of OmpF and OmpC is a direct consequence of the cellular level of the active form of OmpR.

A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation.

Transcription of the genes that encode the major outer membrane porin proteins OmpF and OmpC of Escherichia coli is regulated in response to changes in medium osmolarity by EnvZ and OmpR. EnvZ functions to sense environmental conditions and to relay this information to the DNA-binding protein OmpR. We have used a truncated EnvZ protein (EnvZ115), which is defective in sensory function but able to communicate with OmpR, to study the biochemical interactions between these two proteins and their effects on transcription from the ompF promoter. We show that purified EnvZ115 can phosphorylate OmpR in the presence of ATP. In addition, EnvZ115 stimulates the ability of OmpR to activate ompF transcription in vitro. Using antibodies specific for EnvZ, we have purified the wild-type protein and have shown that it is also an OmpR kinase. These results provide a prokaryotic example of a transmembrane sensory protein that functions as a protein kinase and suggest a mechanism by which EnvZ communicates with OmpR in signal transduction.

A distant upstream site involved in the negative regulation of the Escherichia coli ompF gene.

The two-component regulatory system, OmpR-EnvZ, of Escherichia coli K-12 regulates the expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein which acts as both an activator and a repressor to control ompF transcription. In this article, we describe a new OmpR-binding site that is located between 384 to 351 bp upstream from the ompF start point of transcription. Inactivation of this site by insertion of a 22-bp fragment prevents the repression of ompF expression conferred by the dominant negative mutation, envZ473. On the basis of the location of this binding site, the presence of bent DNA in the ompF regulatory region (T. Mizuno, Gene 54:57-64, 1987), and the fact that mutations altering integration host factor result in constitutive ompF expression (P. Tsui, V. Helu, and M. Freundlich, J. Bacteriol. 170:4950-4953, 1988), we propose that the negative regulation of ompF involves a DNA loop structure.

Staffing levels in endoscopy units.

Staffing the endoscopy area has become increasingly complex. Growth in procedure volumes, changes in technology, and the application of endoscopy in the diagnosis and treatment of disease contribute to the complexities. The manager must deal with these changes, maintain costs, and still provide adequate staffing to ensure patient safety and quality care. The purpose of this article is to present the results of a laboratory manager survey conducted in 1990. Of 51 laboratory managers who responded, those who rated their laboratories to be adequately staffed averaged 4.2 hr per procedure. The survey results may be useful to laboratory managers seeking to calculate staff needs in a typical endoscopy area.

Signal transduction in bacteria: kinases that control gene expression.

A new paradigm, termed two-component regulatory systems, is emerging from the study of signal transduction in bacteria. A simple example of such a system is provided by the Omp regulon of Escherichia coli. This regulon, which controls the expression of the major outer membrane porin proteins OmpF and OmpC in response to changes in osmolarity, includes the inner membrane protein EnvZ (a receptor kinase) and the DNA-binding protein OmpR (a transcriptional activator). Although we do not know what "ligand" is sensed in the Omp system, we can trace the signal transduction pathway from the receptor at the cell surface directly to regulatory sequences within the DNA. Perhaps signal transduction in bacteria can serve as a simple archetype for understanding certain functions performed by receptor kinases and phosphorylated DNA-binding proteins in higher organisms.

Phosphorylation stimulates the cooperative DNA-binding properties of the transcription factor OmpR.

The two-component regulatory proteins OmpR and EnvZ of Escherichia coli K-12 regulate expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein that is involved in both the positive and negative control of ompF transcription. EnvZ is a histidine kinase that phosphorylates OmpR in response to environmental signals. We used DNA migration retardation analysis to examine the interactions of OmpR and the phosphorylated form of OmpR (OmpR-P) with the regulatory region immediately upstream of the ompF promoter. Our results indicate that the binding of OmpR to this regulatory region is cooperative and that phosphorylation significantly stimulates these cooperative interactions. Moreover, although phosphorylation increases the intrinsic binding of OmpR to a single OmpR-binding site, the primary role of phosphorylation in ompF regulation is to facilitate cooperative interactions between OmpR molecules bound at adjacent sites. Based on these results, we propose a model to explain how the phosphorylation of OmpR could stimulate the occupancy of specific sites in the ompF regulatory region, thereby resulting in the activation or repression of ompF transcription under the appropriate environmental conditions.

Suppression of ctc promoter mutations in Bacillus subtilis.

Transcription from the Bacillus subtilis ctc promoter is induced as cells enter stationary phase under conditions in which the enzymes of the tricarboxylic acid cycle are repressed. This transcription requires the presence of a secondary form of RNA polymerase, E sigma B, that is found in exponentially growing cells and in early-stationary-phase cells. Starting with a defective ctc promoter that had either a base substitution at position -15 or a base substitution at position -36, we were able to identify four independent second-site mutations within these mutated promoters that suppressed the effect of the original mutations and thereby restored function to the ctc promoter. Three of these mutated promoters had an additional base substitution(s) at positions -5, -9, or both -5 and -9 that enhanced their utilization in vivo by E sigma B, whereas one of the promoters had a single-base-pair deletion in the -15 region that placed it under a completely different form of regulation than that of the wild-type ctc promoter. In addition to mutations in the ctc promoter region, we also isolated three classes of mutants that exhibited increased ctc expression. The effects of the mutations in these strains were not allele specific, since they increased expression from both mutant and wild-type ctc promoters. One class of mutants which affected expression from the ctc promoter carried mutations that blocked the activity of the tricarboxylic acid cycle. A second class of mutations mapped near cysA and was unable to sporulate. Three-factor transformation crosses and complementation analysis indicated that one of these mutations was an allele of spo0H. The third class of mutations is closely linked to dal and may define a regulatory gene for sigB, the sigma B structural gene.