Development of bacterial artificial chromosome library resources for parasitoid Hymenoptera (Nasonia vitripennis and Nasonia giraulti: Pteromalidae).

The species of the genus Nasonia possess qualities that make them excellent candidates for genetic and genomic studies. To increase the wealth of genomic resources for the genus we constructed publicly available bacterial artificial chromosome (BAC) libraries for Nasonia vitripennis and Nasonia giraulti. Libraries have 36 864 clones each, empty-vector contents of approximately 2% and average insert sizes of 113.1 and 97.7 Kb, respectively, representing 12 and 11 genome equivalents. The N. vitripennis library was used for genome sequence assembly and in efforts at positional cloning of a developmental gene. The genome assembly of N. vitripennis is currently composed on 6181 un-joined scaffolds. These BAC libraries can be used to identify and close regions between scaffolds of the genome assemblies of both species.

Characterization of a human and mouse tetrapyrrole-binding protein.

The cDNA for p22HBP has been cloned from human and mouse, and the protein expressed, purified, and characterized. Both mouse and human proteins bind heme and porphyrins with micromolar K(d)s, are highly homologous, monomeric, and soluble, and have a cytoplasmic location. The proteins bind metalloporphyrins, free porphyrins, and N-methylprotoporphyrin with similar affinities, and mutations of a selected set of putative metal ligating residues did not have any significant effect on the measured K(d)s. That the presence or absence of metal in the porphyrin has no effect on the binding constants and the observation that the EPR signal for heme does not change upon binding to the protein strongly suggest that p22HBP is a generic tetrapyrrole-binding protein rather than a dedicated heme-binding protein. A role for p22HBP in cellular porphyrin metabolism is discussed.

Genes expressed during early stages of rice infection with the rice blast fungus Magnaporthe grisea.

summary A system-wide approach was adopted to further elucidate mechanisms regulating disease outcome between rice and the fungal pathogen Magnaporthe grisea. First, a cDNA library was constructed from M. grisea infected rice at 48 h post-inoculation. The 5' end-sequencing of 619 randomly selected clones revealed 359 expressed sequence tags (ESTs) that had not previously been described. A total of 124 from 260 ESTs with high and moderate similarity scores, based on BlastX, were organized into categories according to their putative function. The largest category of sequences (21%) contained stress or defence response genes. Eleven per cent of identified ESTs were redundant. In a second approach, differential hybridization analysis of the cDNA library using high-density filters resulted in the identification of novel genes and previously characterized M. grisea genes, including several that had previously been implicated in the infection process. A survey of up-regulated cDNA clones revealed clone 29003, which corresponded to the rice peroxidase POX22.3. This gene is known to be expressed in rice upon infection with Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen. Importantly, this approach demonstrates the utility of gene discovery, through ESTs, for revealing novel genes in addition to those previously characterized as being potentially implicated in host-pathogen interactions.

Physical map and organization of chromosome 7 in the rice blast fungus, Magnaporthe grisea.

The rice blast fungus Magnaporthe grisea is a highly destructive plant pathogen and one of the most important for studying various aspects of host-plant interactions. It has been widely adopted as a model organism because it is ideally suited for genetic and biological studies. To facilitate map-based cloning, chromosome walking, and genome organization studies of M. grisea, a complete physical map of chromosome 7 was constructed using a large-insert (130 kb) bacterial artificial chromosome (BAC) library. Using 147 chromosome 7-specific single-copy BAC clones and 20 RFLP markers on chromosome 7, 625 BAC clones were identified by hybridization. BAC clones were digested with HindIII, and fragments were size separated on analytical agarose gels to create DNA fingerprints. Hybridization contigs were constructed using a random cost algorithm, whereas fingerprinting contigs were constructed using the software package FPC. Results from both methods were generally in agreement, but numerous anomalies were observed. The combined data produced five robust anchored contigs after gap closure by chromosomal walking. The genetic and physical maps agreed closely. The final physical map was estimated to cover >95% of the 4.2 Mb of chromosome 7. Based on the contig maps, a minimum BAC tile containing 42 BAC clones was created, and organization of repetitive elements and expressed genes of the chromosome was investigated.

Inhibition of imidazole-induced tyrosinase activity by estradiol and estriol in cultured B16/C3 melanoma cells.

The effect of estrogens on tyrosinase (EC 1.14.18.1) activity was studied in B16/C3 melanoma cultures. Estradiol, estriol, and other related steroids failed to influence tyrosinase activity when added to the medium of proliferating cultures. Imidazole (10 mM), on the other hand, induced the activity of that enzyme 3-fold, as reported previously. Estradiol and estriol blocked imidazole induction, however, unlike the other estrogenic compounds. The blockade occurred within 15 min of hormone addition and was reversible. Dose-response studies revealed that the maximal estradiol effect occurred at 0.75 nM and the half-maximal effect occurred at 0.5 nM. Estriol was more potent, with the maximal blockade occurring at approximately 0.5 nM and half-maximal effect at 0.25 nM. The induction of tyrosinase by imidazole and the blockade of this induction by estradiol and estriol could not be demonstrated in broken cell preparations, suggesting that direct enzyme activation-inactivation was not involved. Studies utilizing inhibitors of protein and RNA synthesis suggest that this effect is mediated at a pre-translational level and is independent of mRNA destabilization.

Local anesthesia.

In summary, many surgical procedures may be safely and comfortably performed utilizing regional anesthesia if only a few guidelines are followed as to choice and usage of local anesthetics. The success of a regional block will always be dependent upon correct needle placement by an experienced physician with good technical skills. However, the safety of the patient is not solely a function of experience. Modern local anesthetic preparations are reliable enough and simple enough to use that all physicians should be capable of achieving optimal patient safety at all times. If placed in a position which seems to require unfamiliar knowledge or expertise, the practitioner need only seek a consultant anesthesiologist for assistance. Plastic surgery is recognized as a specialty that frequently utilizes local anesthetics for office and outpatient procedures. The manner in which these drugs are used or abused determines their clinical reputation as well as that of the physician. It is important to promote a correct understanding of local anesthetic compounds, not only among ourselves as physicians, but also among our patients, who are becoming ever more knowledgeable of medical practice as time goes on.

Clinical comparison of midazolam hydrochloride and midazolam maleate for anesthesia induction.

The current study was undertaken to determine whether the same doses (mg/kg) of 5 mg/ml of midazolam hydrochloride and 2.5 mg/ml of midazolam maleate are required for the induction of anesthesia. Midazolam hydrochloride and midazolam maleate were compared in a prospective double-blind fashion, in which both cardiopulmonary and sedative effects were measured in 12 patients who required repeated anesthesia for serial gynecologic radium insertions. The results showed no significant differences in clinical activity between midazolam maleate and the newer preparation, midazolam hydrochloride. Time of onset, recovery time, lack of venous irritation, and stability of cardiopulmonary variables when using the hydrochloride were essentially the same with the maleate.

Temperature-dependent expression of virulence genes in Shigella species.

The pathogenicity of Shigella spp. involves the ability of the bacteria to penetrate and replicate within the epithelial cells of the large intestine. Model systems for examining the virulence of shigellae employ Henle intestinal epithelial cells in tissue culture and an in vivo assay for virulence in guinea pig eyes (Sereny test). Using these systems, we studied the genetic and physiological bases for the ability of shigellae to invade epithelial cells. We found that expression of virulence in Shigella spp. is dependent on the temperature at which the bacteria are grown. When grown at 37 degrees C, strains of Shigella flexneri 2a, Shigella sonnei, and Shigella dysenteriae 1 were fully virulent and invaded Henle cells. They also produced keratoconjunctivitis in guinea pigs. When grown at 30 degrees C, the bacteria neither penetrated Henle cells nor produced conjunctivitis in the Sereny test and were phenotypically avirulent. Strains grown at 33 degrees C were only partially invasive in the Henle assay, whereas strains grown at 35 degrees C were as invasive as strains grown at 37 degrees C. Using the Henle cell assay, we determined that the loss of ability to penetrate epithelial cells was completely reversed by shifting the growth temperature from 30 to 37 degrees C. The percentage of Henle cells invaded by bacteria increased with increasing time of growth at 37 degrees C. Restoration of invasiveness after growth at 30 degrees C required protein synthesis. When shigellae were grown at 30 degrees C and shifted to 37 degrees C for 2 h in the presence of chloramphenicol, the bacteria remained noninvasive. Similarly treated bacteria grown at 37 degrees C were still invasive. These results suggested that expression of one or more genes required for virulence of Shigella spp. are subject to regulation by growth temperature.

Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid.

In this study, we examined the relationship between the virulence of Shigella flexneri 2a and the ability of strains of S. flexneri 2a to absorb Congo red. Spontaneous nonpigmented (i.e., unable to bind Congo red [Pcr-]) derivatives of a virulent, pigmented (Pcr+) strain of S. flexneri 2a were isolated and assayed for virulence as determined by their ability to invade epithelial cells. All Pcr- mutants examined lost the ability to invade epithelial cells and were thus avirulent. Agarose gel electrophoresis of plasmid DNA from these avirulent, Pcr- mutants showed that the majority of these strains had lost a plasmid band corresponding to a virulence-associated plasmid, pSf2a140. In many of the mutants, concomitant loss of pigmentation, virulence, and pSf2a140 was accompanied by the appearance of a new plasmid, smaller than pSf2a140. We believe these new plasmids to be deletion derivatives of pSf2a140 and that loss of pigmentation and loss of virulence are associated with deletions in pSf2a140. We transduced Pcr- mutants to Pcr+ and isolated transductants which suppressed the Pcr- phenotype. None of the Pcr+ transductants regained the ability to invade epithelial cells. Several suppressors of the Pcr- phenotype were identified as mutations in cell wall biosynthesis. These results support our belief that although pigmentation is usually associated with virulence, genetic determinants unrelated to virulence can also affect the ability of the cell to bind Congo red. Therefore, the ability of S. flexneri 2a to bind Congo red does not necessarily imply the ability to invade epithelial cells. However, loss of ability to bind Congo red is accompanied by loss of virulence.