One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD-paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single-photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two-photon fs pulses in the range of 780-840 nm. Viability in whole-mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two-photon activation of paGFP and the use of a paGFP-fusion protein in investigations of whole organisms.     

Isolation and molecular characterization of LVP1 lipolysis activating peptide from scorpion Buthus occitanus tunetanus

LVP1, a novel protein inducing lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 1% of crude venom proteins, with pHi approximately 6 and molecular mass of 16170 Da. In contrast to well-characterized scorpion toxins, reduction and alkylation of LVP1 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP1 have a respective molecular mass of 8877 and 8807 Da as determined by mass spectrometry. The N-terminal and some internal peptide sequences of LVP1alpha and beta were determined by Edman degradation. The full amino acid sequences of both chains were deduced from nucleotide sequences of the corresponding cDNAs prepared based on peptide sequences and the 3' and 5' RACE methodologies. LVP1alpha and beta cDNAs encode a signal peptide of 22 residues and a mature peptide of 69 and 73 residues, respectively. Each mature peptide contains seven cysteines, which are compatible with an interchain disulfide bridge. The cDNA deduced protein structures share a high similarity with those of some Na+ channel scorpion toxins. LVP1 was not toxic to mice after intracerebro-ventricular injection. LVP1 stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 of approximately 1+0.5 microg/ml. LVP1 subunits did not display any lipolytic activity. As previously described for venom, beta adrenergic receptor (beta AR) antagonists interfere with LVP1 activity. Furthermore, it is shown that LVP1 competes with [3H]-CGP 12177 (beta1/beta2 antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7) M. These results demonstrate the existence of a new type of scorpion venom nontoxic peptides that are structurally related to Na+ channel toxins but can exert a distinct biological activity on adipocyte lipolysis through a beta-type adrenoreceptor pathway.     

Karyometric analysis of actinic damage in unexposed and sun-exposed skin and in actinic keratoses in untreated individuals

OBJECTIVE: To assess the ability of karyometric analysis to demonstrate progression of actinic damage as a function of sun exposure in individuals with actinic keratoses (AKs) and to evaluate the stability of that assessment over a 3-month period. STUDY DESIGN: Biopsies from subjects with AKs were obtained from unexposed skin, sun-exposed skin and AK lesions. Subjects used an SPF 50 sunscreen, and 3 months later additional biopsies were taken from sun-exposed and AK sites. A total of 13,300 nuclei were recorded from 31 subjects. RESULTS: Measures of nuclear abnormality (NA) and effects of sun damage based on discriminant function (DF) scores were derived. Actinic damage levels varied significantly across biopsy site, demonstrating the method's sensitivity. Accrual of actinic damage was demonstrated in sun-exposed skin and AK lesions when all nuclei were examined over 3 months but only for sun-exposed skin when the worst-damaged nuclei were examined. This suggests a ceiling effect of nuclear damage in the progression to abnormality. Within-subject variability was similar for both NA and DF when all nuclei were considered. Among the worst-damaged nuclei (as defined by high DF), DF showed lower within-case variability than NA, perhaps due to a reduction in nuclear heterogeneity. CONCLUSION: Karyometry's ability to detect subtle levels of actinic damage in nuclear chromatin patterns may make it useful in screening agents for possible use in cancer chemoprevention.     

Lessons learned from a regional strategy for resource allocation

Two qualitative case studies focus on the allocation of CDC funds distributed during 2002 for bioterrorism preparedness in two Texas public health regions (each as populous and complex as many states). Lessons learned are presented for public health officials and others who work to build essential public health services and security for our nation. The first lesson is that personal relationships are the cornerstone of preparedness. A major lesson is that a regional strategy to manage funds may be more effective than allocating funds on a per capita basis. One regional director required every local department to complete a strategic plan as a basis for proportional allocation of the funds. Control of communicable diseases was a central component of the planning. Some funds were kept at the regional level to provide epidemiology services, computer software, equipment, and training for the entire region. Confirmation of the value of this regional strategy was expressed by local public health and emergency management officials in a focus group 1 year after the strategy had been implemented. The group members also pointed out the need to streamline the planning process, provide up-to-date computer networks, and receive more than minimal communication. This regional strategy can be viewed from the perspective of adaptive leadership, defined as activities to bring about constructive change, which also can be used to analyze other difficult areas of preparedness.     

Comprehensive surgical treatment of migraine headaches

The purpose of this study was to investigate the efficacy of surgical deactivation of migraine headache trigger sites. Of 125 patients diagnosed with migraine headaches, 100 were randomly assigned to the treatment group and 25 served as controls, with 4:1 allocation. Patients in the treatment group were injected with botulinum toxin A for identification of trigger sites. Eighty-nine patients who noted improvement in their migraine headaches for 4 weeks underwent surgery. Eighty-two of the 89 patients (92 percent) in the treatment group who completed the study demonstrated at least 50 percent reduction in migraine headache frequency, duration, or intensity compared with the baseline data; 31 (35 percent) reported elimination and 51 (57 percent) experienced improvement over a mean follow-up period of 396 days. In comparison, three of 19 control patients (15.8 percent) recorded reduction in migraine headaches during the 1-year follow-up (p < 0.001), and no patients observed elimination. All variables for the treatment group improved significantly when compared with the baseline data and the control group, including the Migraine-Specific Questionnaire, the Migraine Disability Assessment score, and the Short Form-36 Health Survey. The mean annualized cost of migraine care for the treatment group (925 dollars) was reduced significantly compared with the baseline expense (7612 dollars) and the control group (5530 dollars) (p < 0.001). The mean monthly number of days lost from work for the treatment group (1.2) was reduced significantly compared with the baseline data (4.41) and the control group (4.4) (p = 0.003). The common adverse effects related to injection of botulinum toxin A included discomfort at the injection site in 27 patients after 227 injections (12 percent), temple hollowing in 19 of 82 patients (23 percent), neck weakness in 15 of 55 patients (27 percent), and eyelid ptosis in nine patients (10 percent). The common complications of surgical treatment were temporary dryness of the nose in 12 of 62 patients who underwent septum and turbinate surgery (19.4 percent), rhinorrhea in 11 (17.7 percent), intense scalp itching in seven of 80 patients who underwent forehead surgery (8.8 percent), and minor hair loss in five (6.3 percent). Surgical deactivation of migraine trigger sites can eliminate or significantly reduce migraine symptoms. Additional studies are necessary to clarify the mechanism of action and to determine the long-term results.     

Basic amino acids in a distinct subset of signal peptides promote interaction with the signal recognition particle

Previous studies have demonstrated that signal peptides bind to the signal recognition particle (SRP) primarily via hydrophobic interactions with the 54-kDa protein subunit. The crystal structure of the conserved SRP ribonucleoprotein core, however, raised the surprising possibility that electrostatic interactions between basic amino acids in signal peptides and the phosphate backbone of SRP RNA may also play a role in signal sequence recognition. To test this possibility we examined the degree to which basic amino acids in a signal peptide influence the targeting of two Escherichia coli proteins, maltose binding protein and OmpA. Whereas both proteins are normally targeted to the inner membrane by SecB, we found that replacement of their native signal peptides with another moderately hydrophobic but unusually basic signal peptide (DeltaEspP) rerouted them into the SRP pathway. Reduction in either the net positive charge or the hydrophobicity of the DeltaEspP signal peptide decreased the effectiveness of SRP recognition. A high degree of hydrophobicity, however, compensated for the loss of basic residues and restored SRP binding. Taken together, the data suggest that the formation of salt bridges between SRP RNA and basic amino acids facilitates the binding of a distinct subset of signal peptides whose hydrophobicity falls slightly below a threshold level.