The Cryptococcus neoformans gene DHA1 encodes an antigen that elicits a delayed-type hypersensitivity reaction in immune mice.

When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5' rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.

Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens.

Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis. A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A. tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia. This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia. Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy. A transformation frequency of 1 per 10(5) arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium: fungus ratio. This approach requires no special equipment that might complicate biocontainment. Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure. A. tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C. immitis and perhaps other fungi of medical importance.

Floral determination and expression of floral regulatory genes in Arabidopsis.

The expression of the floral regulators LEAFY, APETALA1 and AGAMOUS-LIKE8 was examined during light treatments that induced flowering in Arabidopsis, and was compared to time points at which floral determination occurred. Extension of an 8-hour day by either continuous red- or far-red-enriched light induced LEAFY and AGAMOUS-LIKE8 expression within 4 hours. The 4 hours of additional light was sufficient for floral determination only in the far-red-enriched conditions, while 12-16 hours of additional light was required for floral determination in the red-enriched conditions. These results indicate that the induction of floral regulatory genes and induction of flower formation can be uncoupled under certain circumstances. Expression of LEAFY and AGAMOUS-LIKE8 in the shoot apex at the time of floral determination is also consistent with genetic data indicating that these genes are involved in the first steps of the transition from vegetative to reproductive development. In contrast to LEAFY and AGAMOUS-LIKE8, APETALA1 expression was first observed 16 hours after the start of photoinduction. Since this time point was always after floral determination, APETALA1 is an indicator of floral determination.

CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.

An albino mutant designated cla1-1 (for "cloroplastos alterados', or "altered chloroplasts') has been isolated from a T-DNA-generated library of Arabidopsis thaliana. In cla1-1 plants, chloroplast development is arrested at an early stage. cla1-1 plants behave like wild-type in their capacity to etiolate and produce anthocyanins indicating that the light signal transduction pathway seems to be unaffected. Genetic and molecular analyses show that the disruption of a single gene, CLA1, by the T-DNA insertion is responsible for the mutant phenotype. RNA expression patterns indicate that CLA1 is positively regulated by light and that it has different effects on the steady-state RNA levels of some nuclear- and chloroplast-encoded photosynthetic genes. Although the specific function of the CLA1 gene is still unknown, it encodes a novel protein conserved in evolution between photosynthetic bacteria and plants which is essential for chloroplast development in Arabidopsis.

A characterization of the MADS-box gene family in maize.

Studies on distantly related dicot plant species have identified homeotic genes that specify floral meristem identity and determine the fate of floral organ primordia. Most of these genes belong to a family characterized by the presence of a structural motif, the MADS-box, which encodes a protein domain with DNA-binding properties. As part of an effort to understand how such genes may have been recruited during the evolution of flowers with different organ types such as those found in maize, two members of this gene family in maize, ZAG1 and ZAG2, have been characterized previously. Here, the isolation and characterization of four new members of this gene family, designated ZAP1, ZAG3, ZAG4 and ZAG5, are described and the genetic map position of these and 28 additional maize MADS-box genes is determined. The first new member of this family appears to be the Zea mays ortholog of the floral homeotic gene APETALA1 (AP1) and has been designated ZAP1. One of these genes, ZAG4, is unusual in that its deduced protein sequence includes the MADS domain but lacks the K-domain characteristically present in this family of genes. In addition, its copy number and expression varies among different inbreds. A large number of maize MADS-box genes map to duplicated regions of the genome, including one pair characterized here, ZAG3 and ZAG5. These data underscore the complexity of this gene family in maize, and provide the basis for further studies into the regulation of floral organ morphogenesis among the grasses.

The Arabidopsis AGL8 MADS box gene is expressed in inflorescence meristems and is negatively regulated by APETALA1.

MADS box genes encode putative transcription factors that play important roles in plant and animal development. In plants, MADS box genes are involved in the early step of specifying floral meristem identity as well as the later step of determining the fate of floral organ primordia. Here, we describe the isolation and characterization of a new MADS box gene from Arabidopsis, designated AGL8. Although AGL8 RNA does not accumulate during vegetative growth, it accumulates to high levels in the inflorescence apical meristem as well as in the inflorescence stem and cauline leaves. AGL8 RNA is excluded from the young flower primordia that arise on the flanks of the inflorescence meristem but later accumulates in the walls of the developing carpels. The lack of AGL8 RNA in floral meristems is due in part to the action of another MADS box gene, APETALA1, because AGL8 RNA does accumulate in apetala1 mutant flower primordia.

A gene triggering flower formation in Arabidopsis.

In Arabidopsis, the apical shoot meristem produces lateral meristems that develop into either shoots or flowers. The decision to form flowers instead of shoots is mediated by the action of floral-meristem-identity genes, such as APETALA1 (AP1) and LEAFY (LFY), which specify meristem fate. Here we show that transgenic plants which constitutively express the AP1 gene show transformations of apical and lateral shoots into flowers, and that these plants flower much earlier than wild-type plants. These results indicate that AP1 alone can convert infloresence shoot meristems into floral meristems, and that ectopic AP1 expression can dramatically reduce the time to flowering.

Conversion of perianth into reproductive organs by ectopic expression of the tobacco floral homeotic gene NAG1.

Mutations in the AGAMOUS (AG) gene of Arabidopsis thaliana result in the conversion of reproductive organs, stamens and carpels, into perianth organs, sepals and petals. We have isolated and characterized the putative AG gene from Nicotiana tabacum, NAG1, whose deduced protein product shares 73% identical amino acid residues with the Arabidopsis AG gene product. RNA tissue in situ hybridizations show that NAG1 RNA accumulates early in tobacco flower development in the region of the floral meristem that will later give rise to stamens and carpels. Ectopic expression of NAG1 in transgenic tobacco plants results in a conversion of sepals and petals into carpels and stamens, respectively, indicating that NAG1 is sufficient to convert perianth into reproductive floral organs.

Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS.

Recent genetic and molecular studies in Arabidopsis and Antirrhinum suggest that mechanisms controlling floral development are well conserved among dicotyledonous species. To assess whether similar mechanisms also operate in more distantly related monocotyledonous species, we have begun to clone homologs of Arabidopsis floral genes from maize. Here we report the characterization of two genes, designated ZAG1 and ZAG2 (for Zea AG), that were cloned from a maize inflorescence cDNA library by low stringency hybridization with the AGAMOUS (AG) cDNA from Arabidopsis. ZAG1 encodes a putative polypeptide of 286 amino acids having 61% identity with the AGAMOUS (AG) protein. Through a stretch of 56 amino acids, constituting the MADS domain, the two proteins are identical except for two conservative amino acid substitutions. The ZAG2 protein is less similar to AG, with 49% identity overall and substantially less similarity than ZAG1 outside the well-conserved MADS domain. Like AG, ZAG1 RNA accumulates early in stamen and carpel primordia. In contrast, ZAG2 expression begins later and is restricted to developing carpels. Hybridization to genomic DNA with the full-length ZAG1 cDNA under moderately stringent conditions indicated the presence of a large family of related genes. Mapping data using maize recombinant inbreds placed ZAG1 and ZAG2 near two loci that are known to affect maize flower development, Polytypic ear (Pt) and Tassel seed4 (Ts4), respectively. The ZAG1 protein from in vitro translations binds to a consensus target site that is recognized by the AG protein. These data suggest that maize contains a homolog of the Arabidopsis floral identity gene AG and that this gene is conserved in sequence and function.

Syringe liposculpture revisited.

Syringe liposculpture was introduced by Fournier almost a decade ago. Recent advances have improved the technique, making it easier for the surgeon to use. To determine the efficacy of the syringe in reducing morbidity, groups of patients who had between 1200 and 1500 mL of adipose tissue removed were evaluated using different infiltration and power source (syringe vs machine) regimens. There was less blood lost when the syringe was used. In addition, syringe-treated patients appeared to heal faster, return to work in a shorter period of time, and have less pain. The advantages of the syringe technique are considerable and increase the safety factor of the liposculpture procedure.

Molecular characterization of the Arabidopsis floral homeotic gene APETALA1.

The first step in flower development is the transition of an inflorescence meristem into a floral meristem. Each floral meristem differentiates into a flower consisting of four organ types that occupy precisely defined positions within four concentric whorls. Genetic studies in Arabidopsis thaliana and Antirrhinum majus have identified early-acting genes that determine the identify of the floral meristem, and late-acting genes that determine floral organ identity. In Arabidopsis, at least two genes, APETALA1 and LEAFY, are required for the transition of an influorescence meristem into a floral meristem. We have cloned the APETALA1 gene and here we show that it encodes a putative transcription factor that contains a MADS-domain. APETALA1 RNA is uniformly expressed in young flower primordia, and later becomes localized to sepals and petals. Our results suggest that APETALA1 acts locally to specify the identity of the floral meristem, and to determine sepal and petal development.

Manipulation of flower structure in transgenic tobacco.

Genetic studies suggest that three homeotic functions, designated A, B, and C, act alone and together to specify the fate of floral organ primordia in distantly related dicotyledonous plant species. To test the genetic model, we have generated transgenic tobacco plants that ectopically express the AGAMOUS gene from Brassica napus, which is necessary for the C function. Flowers on the resulting plants showed homeotic transformations of sepals into carpels and petals into stamens. These phenotypes are consistent with predictions from the genetic model, show that expression of AGAMOUS is sufficient to provide ectopic C function, and demonstrate that the structure of flowers can be manipulated in a predictable manner by altering the expression of a single regulatory gene. Furthermore, the generation of the predicted transformations by ectopic expression of the Brassica gene in transgenic tobacco indicates that gene functions are interchangeable between phylogenetically distant species.

A commercial large-vocabulary discrete speech recognition system: DragonDictate.

DragonDictate is currently the only commercially available general-purpose, large-vocabulary speech recognition system. It uses discrete speech and is speaker-dependent, adapting to the speaker's voice and language model with every word. Its acoustic adaptability is based in a three-level phonology and a stochastic model of production. The phonological levels are phonemes, augmented triphones (phonemes-in-context or PICs), and steady-state spectral slices that are concatenated to approximate the spectra of these PICs (phonetic elements or PELs) and thus of words. Production is treated as a hidden Markov process, which the recognizer has to identify from its output, the spoken word. Findings of practical value to speech recognition are presented from research on six European languages.

Minimal suture blepharoplasty: closure of incisions with autologous fibrin glue.

Blepharoplasty incisions can be cloned safely with autologous fibrin glue. The fibrinogen, prepared either from a whole-blood or plasmapheresis source, is mixed with commercially available thrombin to form a seal that is both hemostatic and adhesive. The complication rate is low and primarily due to technical factors in the initial cases. When compared with standard suture techniques, the incidence of minor problems such as milia formation was much lower. In select cases, the technique of using fibrin glue and a minimal number of sutures may be useful as an alternative method of wound closure in blepharoplasty patients.

Treatment of glabellar frown lines using silicone implants.

The presence of deep glabellar frown lines will make even a young patient appear old and angry. This is particularly true when the sole sign of aging is prominent wrinkling between the eyes. Previous attempts at correction including direct excision, autologous fat transfers, collagen injections, and coronal lift have resulted only in transient improvement or noticeable scars and considerable morbidity. The current technique is a safe, effective, and simple alternative to soften the frown lines and decrease glabellar area deficiencies. Twenty-five patients have been treated with good results by using a shield-shaped silicone implant. The muscles do not adhere to the implant, resulting in significant improvement in skin creases, both at rest and with facial animation. Complications are minimal with a rapid return of patients to their normal activities. This simple technique softens and corrects a noticeable deficiency with a high degree of patient satisfaction and minimal morbidity.

Closure of blepharoplasty incisions with autologous fibrin glue.

Autologous fibrin glue was prepared from individual patients and used as a surgical adhesive. Sixteen patients undergoing elective eyelid operations were studied. The fibrinogen was prepared from autologous blood by a cryoprecipitate technique. When mixed with commercially available thrombin, a fibrin clot develops with sufficient adhesive strength that the need for extensive suturing is obviated. Complications were few, and due to technical factors in the initial cases, all patients were followed up for at least 1 year. Problems associated with suture closure of wounds (eg, cysts, granulomas, milia) were not seen. The fibrin glue not only sealed the wound but also acted as a hemostatic agent. The autologous preparation is superior to commercial products since it avoids the problem of transfusion-transmitted disease. The fibrin glue and minimal suture technique is an alternative to eyelid incision closure and may be useful in many other types of operative procedures.

Blood and fluid replacement in major liposuction procedures.

Major liposuction procedures require considerable fluid resuscitation to make them safe. A simple fluid replacement formula is presented that will estimate crystalloid and blood losses. It takes into account the daily fluid requirement, volume extracted, and the multiplicity of areas treated. Removal of 1500 mL required single-unit autotransfusion while amounts greater than 2500 mL necessitated a two-unit autotransfusion. These figures must be tempered by the surgeon's clinical assessment of each patient. The keys to successful and safe management of the liposuction patient are proper preoperative preparation and adequate fluid replacement.