Single centrosome manipulation reveals its electric charge and associated dynamic structure.

The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 +/- 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 10(3) electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca(2+), over the thermally induced dynamic structure of the centrosome.

Structure and function of the centriole in animal cells: progress and questions.

The centriole is a well-recognized, yet poorly understood, organelle present in many eukaryotic cells. Despite excellent electron-microscopic descriptions of its basic triplet microtubule structure, almost nothing is known of its specific molecular components. Here, Bodo Lange and Keith Gull survey centriole structure, duplication and maturation within the cell cycle and focus attention on the possible roles and function of centrioles as components of the centrosome in animal cells.

A molecular marker for centriole maturation in the mammalian cell cycle.

The centriole pair in animals shows duplication and structural maturation at specific cell cycle points. In G1, a cell has two centrioles. One of the centrioles is mature and was generated at least two cell cycles ago. The other centriole was produced in the previous cell cycle and is immature. Both centrioles then nucleate one procentriole each which subsequently elongate to full-length centrioles, usually in S or G2 phase. However, the point in the cell cycle at which maturation of the immature centriole occurs is open to question. Furthermore, the molecular events underlying this process are entirely unknown. Here, using monoclonal and polyclonal antibody approaches, we describe for the first time a molecular marker which localizes exclusively to one centriole of the centriolar pair and provides biochemical evidence that the two centrioles are different. Moreover, this 96-kD protein, which we name Cenexin (derived from the Latin, senex for "old man," and Cenexin for centriole) defines very precisely the mature centriole of a pair and is acquired by the immature centriole at the G2/M transition in prophase. Thus the acquisition of Cenexin marks the functional maturation of the centriole and may indicate a change in centriolar potential such as its ability to act as a basal body for axoneme development or as a congregating site for microtubule-organizing material.

bHLH093/NFL and bHLH061 are required for apical meristem function in Arabidopsis thaliana.

The basic Helix-Loop-Helix (bHLH) transcription factors SCREAM/ICE1 and SCREAM2 have well-characterized roles in the terminal differentiation of stomatal guard cells in Arabidopsis thaliana. Here we report on the characterization of the functional roles of the remaining members of sub-group IIIB, bHLH093 and bHLH061. The bhlh093/bhlh061 double mutant failed to produce a primary inflorescence shoot and displayed greater phenotypic severity than bhlh093 and bhlh061 single mutants. An ultrastructural investigation revealed structural defects that develop in tissues surrounding the meristem prior to inflorescence emergence. The transition to flowering was restored in bhlh093/bhlh061 with the application of gibberellin to the apex. We also demonstrate that gibberellin application alleviates structural defects that develop in tissues surrounding the meristem and restore meristem activity. Furthermore, the bhlh093/bhlh061 double mutant was affected by delayed flowering, and the severity of the phenotype correlated with photoperiod and light intensity. Our results indicate that bHLH093 and bHLH061 function in the gibberellin-mediated establishment of functional apical meristems during the transition from vegetative to reproductive growth.

Genetic engineering of essential oil production in mint.

New approaches directed to unraveling monoterpene metabolism and secretion and recent progress in transformation protocols have set the stage for the systematic genetic engineering of essential oil production. This article focuses on specific strategies to improve the quality and quantity of mint essential oils.

Generation and Functional Evaluation of Designer Monoterpene Synthases.

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes.

Elicitor-Induced Spruce Stress Lignin (Structural Similarity to Early Developmental Lignins).

Suspension cultures of Picea abies (L.) Karst released polymeric material into the culture medium when treated with an elicitor preparation from the spruce needle pathogen Rhizosphaera kalkhoffii. The presence of lignin (about 35%, w/w) was demonstrated by phloroglucinol/HCI reactivity and quantitation with thioglycolic acid. Carbohydrate (about 14%, w/w) and protein (about 32%, w/w) were also detected. Amino acid analysis revealed that hydroxyproline and proline predominated. Thioacidolysis and subsequent Raney nickel desulfurization allowed the analysis of lignin-building units and interunit bonds. Compared with spruce wood lignin, an approximately 20-fold higher relative amount of p-hydroxyphenyl units was determined. A high content of p-hydroxyphenyl units is typical for certain developmental lignins, such as conifer compression wood and middle lamella lignins, as well as all induced cell culture lignins so far analyzed. Cross-linkages of the pinoresinol type ([beta]-[beta]) in the excreted cell culture lignin were markedly increased, whereas [beta]-1 interunit linkages were decreased relative to spruce wood lignin. The amount and nature of cross-linkages were shown to be intermediate between those in wood lignin and in enzymatically prepared lignins. In summary, the elicitor-induced stress lignin was excreted as a lignin-extensin complex that closely resembled early developmental lignins.

Hereditary spastic paraplegia linked to chromosome 15q: Analysis of candidate genes.

We previously reported an extended kindred with autosomal dominant uncomplicated hereditary spastic paraplegia (HSP) and found close linkage between the disorder and microsatellite polymorphisms on chromosome 15q. Multipoint linkage analysis reached a maximum LOD score (10.16) between D15S128 and D15S156, a region that includes genes encoding alpha5 and beta3 subunits of GABAA receptor. Theoretically, abnormal GABA-mediated neurotransmission could produce spasticity and possibly other changes of HSP. We used genetic linkage analysis to evaluate these two HSP candidate genes and observed obligate recombinants for polymorphisms immediately adjacent to (or within untranslated regions of) genes encoding alpha5 and beta3 GABAA receptor subunits. Although these genes are linked tightly to the HSP locus, our findings conclusively exclude these genes from being responsible for HSP in this kindred.

Autosomal dominant, familial spastic paraplegia, type I: clinical and genetic analysis of a large North American family.

"Familial spastic paraplegia" (FSP) refers to clinically and genetically diverse syndromes characterized by insidiously progressive lower extremity spasticity. We evaluated 126 members of a large kindred, including 31 affected subjects, in which FSP was transmitted as a stereotyped, autosomal dominant disorder that showed complete genetic penetrance. Affected subjects developed insidiously progressive gait disturbance between ages 12 and thirty-five. Neurologic examination revealed hyperreflexia and spasticity in the lower extremities, weakness of hip flexion and ankle dorsiflexion, extensor plantar response, diminished vibratory sense in the feet, and pes cavus. Using genetic linkage analysis, we excluded the FSP1 locus on chromosome 14q11.2 as the disease locus in this family. We present the clinical and genetic features of FSP type I, including the age-adjusted risk of developing the disorder in this family.

Communication tendencies of senior dental students.

Although the importance of dentist-patient communication is generally recognized, the dental literature does not specify what interpersonal behaviors can be used as a basis for evaluating dental students' communication skills. A set of behaviors based on clinical observations is described, and an evaluation of 25 senior students' behavioral skills is presented. Students often did not: (1) spend much time orienting patients before beginning treatment; (2) update medical histories; (3) ask patients if they had any questions before beginning treatment; (4) explain the operation and use of equipment; (5) forewarn patients about uncomfortable procedures; (6) attend to signs of patient discomfort; (7) reinforce praiseworthy patient behavior; (8) caution patients about numbness, chewing, and sensitivity; (9) thank patients for their time; and (10) use leading/motivating questions. There is a need to provide students with systematic feedback on their interpersonal behaviors.

The impact of a curriculum change on student attitude and ultimate professional behavior.

The longitudinal effects of curriculum changes on student attitude and the relationship between attitudes as students and behavior as a professionals in practice are explored in this paper. A curriculum change (treatment of handicapped patients) was planned, introduced, and its effects on student attitude and professional behavior monitored. Findings indicate that: (1) exposure to handicapping conditions significantly lowered student humanitarian attitudes about the treatment of handicapped patients, (2) involvement with handicapped, regardless of the type of involvement (i.e. clinical or supervising oral hygiene care), appears to adversely affect humanitarian attitudes of students regarding handicapped patients. (3) student estimation of their future behavior is not a reliable indication of their future behavior, and (4) changes in curriculum should not be evaluated from student attitudes alone.

Management of patients needing antibiotic prophylaxis in a dental education setting.

The management of antibiotic prophylaxed (ABX) patients at an educational institution was evaluated to identify areas for improvement. Management criteria, reflecting guidelines to prevent oral-induced hematogenous microbial seeding, were pretested and applied to 1,225 record entries of eighty-five patients needing ABX for dental treatment between 1991 and 1996. Seven hundred twenty-two of the visits had 857 management or documentation problems, including no documentation indicating whether or not patients premedicated (n = 281); incomplete, insufficient, or repeated treatment (n = 214); and preventive concerns (n = 172), among others. The proportion of providers' patient visits with one or more management problems differed significantly (p < 0.001) by provider type, as did the distribution of problem categories (documentation, treatment, preventive, and scheduling concerns p < 0.001; compliance issues p < 0.005). Fifty-one percent of postgraduates' and 39 percent of faculty's record entries omitted patients' ABX status. Improved documentation, outcome measures, and patient, faculty and student education are indicated.

Preferences for an influences on oral health prevention: perceptions of directors of nursing.

Directors of Nursing (DONs) from 196 of 206 Nebraska long-term-care (LTC) facilities were sent a pre-tested questionnaire. The aim was to assess available on-site dental services, existing oral health education and prevention programs as well as future needs/preferences, and the influence of 10 factors in assessing and maintaining residents' oral health. Of the 196 DONs contacted, 126 (64%) participated. Only 36% of DON responders reported having on-site dental services. DONs indicated a preference for nursing staff (NS) oral health inservice training over other educational and/or programmatic proposals. When asked to select the five most influential factors in assessing and maintaining residents' oral health in their respective facilities, DONs selected resident factors (mean, 2.8) more often than NS factors (mean, 2.2). DONs most often identified residents' (R') ability to perform oral hygiene (n = 99), R' cooperation with OH assistance (n = 98), R' interest in their oral health (n = 83), NS interest in R' dental health (n = 70), and NS time constraints (n = 69).

Staff meetings as perceived by dental assistants.

CDAs generally prefer meetings with these characteristics: organization; written agendas; longer, frequent, and regularly scheduled; compensated attendance; follow-up on progress toward goals/objectives; avoidance of personal and individual-compensation issues; input from staff members, and open discussion; and encouragement and praise. These results should help dentists and staff members to make staff meetings more efficient and effective.