Management of systemic prostate cancer: current algorithm from castration sensitive to castration resistant setting.

In recent years, the advanced knowledge of clinical, biological and molecular features of prostate cancer have led to the introduction of new drugs and have allowed the relocation of old drugs in different settings. In this way, the new concepts of systemic disease arise: high risk or high volume vs. low risk and low volume disease castration sensitive prostate cancer (CSPC), diversifying the use of previously approved drugs (CRPC) and opening new scenarios for sequence therapy. The aim of this review is to integrate new developments into the medical management of systemic prostate cancer.

Trocar-guided trans-vaginal mesh surgery for pelvic organ prolapse: effects on urinary continence and anatomical and functional outcomes. A prospective observational study.

OBJECTIVE: Primary objective of this study was to assess the effects of trocar-guided transvaginal mesh surgery (TVM) on cure and prevention rates for incontinence, without concomitant surgery for Stress Urinary Incontinence (SUI). Our secondary objectives were anatomical outcomes, relief of symptoms and effect on quality of life (QoL). STUDY DESIGN: This prospective observational study evaluated women who underwent TVM for symptomatic stage >2 Pelvic Organ Prolapse (POP). SUI was evaluated objectively using the cough stress test with prolapse reduced. SUI and urge urinary incontinence (UUI) were subjectively evaluated using ICIQ-SF. Anatomical cure was defined as stage <2 at POP-Q. STATISTICAL ANALYSIS: McNemar chi-square test; paired t-test; Mann-Whitney test. RESULTS: Seventy-two patients reached final evaluation (mean follow-up 72 months). In the 40 pre-op continent patients, 34 (85%) remained continent postoperatively and 6 (15%) showed de novo SUI. Only 1 patient chose to undergo subsequent TVT. The number needed to treat was 6 to prevent 1 women developing de novo objective SUI and 39 to prevent 1 woman having to undergo SUI surgery. In the 32 pre-op incontinent patients, 18 (56.3%) became continent postoperatively. Only 1 patient chose to undergo subsequent TVT. UUI was present in 44 patients pre-operatively and 15 (20.8%) post-operatively (1 de novo). Forty-four patients (61.1%) were continent post-operatively for SUI and UUI. We observed a significant improvement in storage, voiding, post-micturition and prolapse-related symptoms. The anatomical cure rate was 87.5% for the anterior compartment and 90.3%.for the apical segment. The apical recurrence was 8.3% in the patients previously hysterectomised, 18.8% in the patients with uterus preservation and 0% in the patients with concomitant hysterectomy. QoL scores improved in all domains except sleep and personal relationships. We observed mesh exposure in 10 patients (13.9%), in 5 of whom it was associated with a concomitant hysterectomy CONCLUSIONS: TVM showed excellent results in terms of continence and can be performed without contemporary anti-incontinence surgery, for both continent and incontinent women. Patients should have pre-operative counselling before POP surgery. For severe uterine prolapse the Perigee™ System should be employed with concomitant hysterectomy because uterus preservation is associated with significantly higher apical recurrence rates.

Mucoactive drugs.

Mucus hypersecretion is a clinical feature of severe respiratory diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Airway mucosal infection and/or inflammation associated with these diseases often gives rise to inflammatory products, including neutrophil-derived DNA and filamentous actin, in addition to bacteria, apoptotic cells and cellular debris, that may collectively increase mucus production and viscosity. Mucoactive agents have been the medication of choice for the treatment of respiratory diseases in which mucus hypersecretion is a clinical complication. The main purpose of mucoactive drugs is to increase the ability to expectorate sputum and/or decrease mucus hypersecretion. Many mucoactive drugs are currently available and can be classified according to their putative mechanism of action. Mucoactive medications include expectorants, mucoregulators, mucolytics and mucokinetics. By developing our understanding of the specific effects of mucoactive agents, we may result in improved therapeutic use of these drugs. The present review provides a summary of the most clinically relevant mucoactive drugs in addition to their potential mechanism of action.

Drought tolerance of selected Eragrostis species correlates with leaf tensile properties.

BACKGROUND AND AIMS: Previous studies on grass leaf tensile properties (behaviour during mechanical stress) have focused on agricultural applications such as resistance to trampling and palatability; no investigations have directly addressed mechanical properties during water stress, and hence these are the subject of this study. METHODS: Critical (lethal) relative water contents were determined for three species of grass in the genus Eragrostis varying in their tolerance to drought. Measurements were taken for leaf tensile strength, elastic modulus, toughness and failure load under different conditions of hydration, and light microscopy and histochemical analyses were undertaken. KEY RESULTS: Leaf tensile strength of fully hydrated leaves for the drought-intolerant E. capensis, the moderately drought-tolerant E. tef and the drought-tolerant E. curvula correlated well with drought tolerance (critical relative water content). Eragrostis curvula had higher tensile strength values than E. tef, which in turn had higher values than E. capensis. Measurements on the drought-tolerant grass E. curvula when fully hydrated and when dried to below its turgor loss point showed that tensile strength, toughness and the elastic modulus all increased under conditions of turgor loss, while the failure load remained unchanged. Additional tests of 100 mm segments along the lamina of E. curvula showed that tensile strength, toughness and the elastic modulus all decreased with distance from the base of the lamina, while again the failure load was unaffected. This decrease in mechanical parameters correlated with a reduction in the size of the vascular bundles and the amount of lignification, as viewed in lamina cross-sections. CONCLUSIONS: The results confirm that leaf mechanical properties are affected by both water status and position along the lamina, and suggest a positive correlation between leaf internal architecture, tensile strength, cell wall chemistry and tolerance to dehydration for grasses.

Zinc ions alter morphology and chitin deposition in an ericoid fungus.

A sterile mycelium PS IV, an ascomycete capable of establishing ericoid mycorrhizas, was used to investigate how zinc ions affect the cellular mechanisms of fungal growth. A significant reduction of the fungal biomass was observed in the presence of millimolar zinc concentrations; this mirrored conspicuous changes in hyphal morphology which led to apical swellings and increased branching in the subapical parts. Specific probes for fluorescence and electron microscopy localised chitin, the main cell wall polysaccharide, on the inner part of the fungal wall and on septa in control specimens. In Zn-treated mycelium, hyphal walls were thicker and a more intense chitin labelling was detected on the transverse walls. A quantitative assay showed a significant increase in the amount of chitin in metal-treated hyphae.

Identification, subcellular localization, and developmental studies of oleosins in the anther of Brassica napus.

mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15-residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS-PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of approximately 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N-terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of lowdensity organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule-filled plastids and lipid-containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low-density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil-body oleosins share common epitopes at the generally non-conserved C-terminal domain with the sporophytic tapetum oleosins.

Oleosin of plant seed oil bodies is correctly targeted to the lipid bodies in transformed yeast.

Yeast (Saccharomyces cerevisiae) has been used extensively as a heterologous eukaryotic system to study the intracellular targeting of proteins to different organelles. The lipid bodies in yeast have not been previously subjected to such studies. These organelles are functionally equivalent to the subcellular storage oil bodies in plant seeds. A plant oil body has a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We tested whether plant oleosin could be correctly targeted to the lipid bodies in transformed yeast. The coding region of a maize (Zea mays L.) oleosin gene was incorporated into yeast high copy and low copy number plasmids in which its expression was under the control of GAL1 promoter. Yeast strains transformed with these plasmids produced oleosin when grown in a medium containing galactose but not glucose. The oleosin produced in yeast had a molecular mass slightly higher than that of the native protein in maize. Oleosin accumulated concomitantly with the storage lipids during growth of the transformed yeast, and it was not secreted. Subcellular fractionation of the cell extracts obtained by two different cell breakage procedures revealed that the oleosin was largely restricted to the lipid bodies. Oleosin apparently did not affect the lipid contents and composition of the transformed yeast lipid bodies but replaced some of the native proteins associated with the organelles. Immunocytochemistry of the transformed yeast cells showed that the oleosin was present mostly on the periphery of the lipid bodies. Oleosin isolated from maize or transformed yeast strain, alone or in the presence of phospholipids or SDS, did not bind to the yeast lipid bodies in vitro. We conclude that plant oleosin is correctly targeted to the lipid bodies in transformed yeast and that yeast may be used as a heterologous system to dissect the intracellular targeting signals in the oleosin.

Detection of numerical aberrations in hematologic neoplasias by fluorescence in situ hybridization.

BACKGROUND AND OBJECTIVE: Over the last 5 years, fluorescence in situ hybridization (FISH) techniques have had an important impact on molecular cytogenetic diagnosis, providing a better understanding of the role of numerical aberrations in hemopoietic neoplasms. The objective of this article is to analyze the clinical applications of FISH in the management of hemopoietic malignancies. EVIDENCE AND INFORMATION SOURCES: The material examined in the present review includes articles and abstracts published in journals covered by the Science Citation Index and Medline, and personal published and unpublished data. STATE OF ART: FISH technology has the advantage of being relatively simple, fast and flexible. Published data and ongoing prospective studies show that, under well-controlled experimental conditions, interphase FISH is more sensitive than conventional metaphase analysis in the detection of numerical abnormalities. Due to the relatively high rate of false positive results, FISH cannot be used for the study of minimal residual disease. However, since molecular strategies for the detection of small-sized aneuploid clones have not been developed yet, FISH represents a useful adjunct to conventional cytogenetics, especially for the quantitation of the size of abnormal clones during the course of the disease and to monitor XX/XY chimerism following sex mis-matched bone marrow transplantation. Different approaches to the study of multiple cell-lineage involvement by chromosome changes have been developed that take advantage of FISH techniques by: a) simultaneous FISH and membrane immunophenotyping of cytologic and histologic preparations; b) two-step analysis based on assessment of the morphology of cells on panoptical stains, with subsequent hybridization and relocation of previously identified cells; c) FISH analysis of enriched cell fractions obtained by cell sorting or by separation of bone marrow cells on a density gradient, and d) study of single hemopoietic colonies grown in semisolid media. PERSPECTIVES: New molecular cytogenetic techniques, such as dual color FISH comparative genomic hybridization, are at hand that will greatly improve the diagnostic power of cytogenetics and make FISH increasingly useful in research laboratories as well as in clinical practice.

Interleukin-3 plus interleukin-6 may improve chromosomal analysis of multiple myeloma: cytologic and cytogenetic evidence in thirty-four patients.

To better define the role of interleukin-3 (IL-3) and IL-6 in the cytogenetic analysis of multiple myeloma (MM), we performed concomitant chromosome and cytologic studies in 34 patients. In each case, 10-30 x 10(6) bone marrow cells were incubated in two independent cultures consisting of conventional cytogenetic medium with and without IL-3 plus IL-6 added for 72 hours. 1-ml aliquots of each culture were aspirated at 24, 48, and 72 hours and exposed to colcemid for 6 hours. Cytospin preparations were then made and mitotic cells were counted and identified as plasma cells or as nonmalignant cells based on their reactivity with an appropriate anti kappa/lambda serum. Slides for conventional cytogenetic analysis were prepared at 72 hours. A greater than two-fold increase of mitotic plasma cells was observed in cytospin preparations from stimulated cultures versus unstimulated cultures in 15 of 34 cases, whereas a less than 2-fold increase, no variation or no mitosis was recorded in 19 cases. Comparison of the number of mitotic plasma cells in stimulated cultures at 24, 48, and 72 hours showed a decreased mitotic activity at 72 hours. Clonal abnormalities were detected by conventional cytogenetic analysis in 19 of 34 cases (55.8%). Recurrent clonal aberrations involved chromosome 13 (4 cases), chromosomes 1p, and 14q (3 cases); chromosomes 3p, 6q, 7q, and 9q (2 cases). We conclude that IL-3 + IL-6 may increase the number of dividing plasma cells in cytogenetic cultures and that a 2-day culture with these cytokines may facilitate the detection of chromosome abnormalities in MM.

Oleosin genes in maize kernels having diverse oil contents are constitutively expressed independent of oil contents. Size and shape of intracellular oil bodies are determined by the oleosins/oils ratio.

In seeds, the subcellular storage oil bodies have a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We used two maize (Zea mays L.) strains having diverse kernel (seed) oil contents to study the effects of varying the oil and oleosin contents on the structure of the oil bodies. Illinois High Oils (IHO, 15% w/w oils) and Illinois Low Oils (ILO, 0.5%) maize kernels were the products of breeding for diverse oil contents for about 100 generations. In both maize strains, although the genes for oil synthesis had apparently been modified drastically, the genes encoding oleosins appeared to be unaltered, as revealed by Southern blot analyses of the three oleosin genes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with immunoblotting of the oleosins. In addition, both strains contained the same three oleosin isoforms of a defined proportion, and both accumulated oils and oleosins coordinately. Oleosins in both strains were restricted to the oil bodies, as shown by analyses of the various subcellular fractions separated by sucrose-density-gradient centrifugation. Electron microscopy of the embryos and the isolated organelles revealed that the oil bodies in IHO were larger and had a spherical shape, whereas those in ILO were smaller and had irregular shapes. We conclude that in seeds, oleosin genes are expressed independent of the oil contents, and the size and shape of the oil bodies are dictated by the ratio of oils to oleosins synthesized during seed maturation. The extensive breeding for diverse oil contents has not altered the apparent mechanism of oil-body synthesis and the occurrence of hetero-dimer or -multimer of oleosin isoforms on the oil bodies.

Isolation of mesophyll and secretory cell protoplasts of the halophyte Ceratostigma plumbaginoides (L.): a comparison of ATPase concentration and activity.

A method is described for isolating mesophyll protoplasts from leaves and secretory cell protoplasts from salt glands of the facultative halophyte, Ceratostigma plumbaginoides (L.). Rates of ATP hydrolysis in both cell types were determined, and levels in secretory cell protoplast preparations were fourfold higher than those in mesophyll protoplast preparations, based on total protein. The rate of ATP hydrolysis was sensitive to azide and vanadate, and stimulated by Triton-X-100. Additionally, immunoblot procedures using an antibody to the plasma membrane H(+)/ATPase was used to compare ATPase levels of the mesophyll and secretory cell protoplasts. Results indicate that secretory cells have a higher concentration of H(+)/ATPase than mesophyll cells, consistent with their putative function in salt glands.

Heterogeneity of lineage involvement by trisomy 8 in myelodysplastic syndrome. A multiparameter analysis combining conventional cytogenetics, DNA in situ hybridization, and bone marrow culture studies.

To better understand the role of trisomy 8 in myelodysplastic syndrome (MDS), we performed a multiparameter analysis combining conventional chromosome studies (CCS), fluorescence in situ hybridization (FISH), and bone marrow (BM) culture studies in two patients with MDS evolving into acute myeloid leukemia (AML). A mosaicism of a cytogenetically normal clone and a clone with trisomy 8 was detected in both patients throughout the course of the disease, a finding confirmed by FISH on BM cells. The relative size of the trisomic clone increased from 52% to 71% (p < 0.0001) and from 53% to 69% (p = 0.001) of all BM cells at the time of the leukemic switch in patients 1 and 2, respectively. Combined FISH and immunophenotyping of BM cells showed involvement of the granulomonocytic lineage in patient 1 and involvement of erythroid cells as well as of the granulomonocytic lineage in patient 2. Only disomic lymphocytes were detected in both patients. FISH on single hemopoietic colonies grown in semisolid media detected trisomic CFU-GM and disomic BFU-E in patient 1, whereas a proportion of CFU-GM and BFU-E deriving from the trisomic clone was detected in patient 2. However, the percent of trisomic colonies was lower than the percent of involved granulomonocyte precursors and involved erythroblasts, as detected by combined FISH and immunophenotyping on fresh BM samples. We have thus shown heterogeneity of lineage involvement by trisomy 8 in MDS undergoing transformation into AML. Although preferential growth of disomic clones may occur in vitro, the finding of an increased size of the trisomic clone at the time of leukemic switch suggests that these cells had proliferative advantage in vivo over cells without trisomy 8.

Atypical chronic lymphocytic leukaemia with t(11;14)(q13;q32): karyotype evolution and prolymphocytic transformation.

In order to define better the cytological and clinical features of atypical B-cell chronic lymphocytic leukaemia (B-CLL) with t(11:14)(q13;q32), sequential morphologic immunological and cytogenetic studies were performed in seven patients belonging to a series of 72 consecutive cases presenting with a diagnosis of CLL or atypical CLL according to the FAB criteria. Cytologic diagnosis in these seven patients with t(11;14) was typical CLL in two cases presenting with < 10% large lymphocytes (LL) and prolymphocytes (PL) and atypical CLL in five cases in which LL and PL comprised between 10% and 55%. The diagnosis was supported by histologic findings on bone marrow biopsy (five cases) or splenectomy specimens (two cases). A progressive increase of peripheral LL and PL was observed, resulting in a switch of FAB diagnosis over a 6-60-month period from typical CLL into atypical CLL in two cases and from atypical CLL into prolymphocytic leukaemia in five cases. Immunophenotyping showed a mature B-cell phenotype with CD19, CD22, CD24 positivity and CD10 negativity in all patients. A bright-staining pattern for surface immunoglobulins (SIg) was detected in 6/7 cases, CD5 positivity in 6/7 cases, and CD23 positivity in 1/7 cases. The FMC-7 monoclonal antibody was positive in > 40% cells in 5/6 cases. Chromosome changes in addition to t(11;14) were seen in five cases; in two cases unbalanced translocations involving the 3q21 chromosome region, resulting in partial trisomy for the long arm of chromosome 3, were detected early in the course of the disease. Karyotype evolution that was associated with disease progression occurred in 3/6 assessable patients. Comparison of these findings with similar data from 65 B-CLL patients without t(11:14) showed that atypical morphology, switch of FAB diagnosis during the course of the disease, and karyotype evolution were more frequently seen in cases with t(11;14) (5/7 v 15/65 cases, P = 0.015, 7/7 v 7/65 cases, P < 0.0001, and 3/6 v 5/45 assessable cases, P = 0.04, respectively). The frequency of positivity for CD23 and bright SIg staining differed significantly in the two groups. It is concluded that t(11;14) identifies a cytologically atypical subset of B-CLL, characterized by frequent cytologic and cytogenetic evolution and by a distinct immunological profile, sharing some biological features with mantle cell lymphoma.

Flow cytometry evaluation of urokinase-type plasminogen activator receptor (UPA-R) in acute myeloid leukemia cells.

The aim of this study was to investigate by flow cytometry the expression of the UPA-R (Urokinase type plasminogen activator receptor-CD87) on the blastic population of AML and ALL patients in order to evaluate whether the presence of this molecule could be associated with peculiar clinical and biologic features of leukemic cells. Five different monoclonal antibodies (MoAbs) (clones: 3B10#; VIM5*; 109#; 68#; 100#) were used in order to detect the distinct forms of this cellular receptor. Cell reactivity varied significantly from case to case, also depending on the MoAb used for the flow cytometry analysis. In brief, 3B10# and VIM5* MoAbs were found to be positive in more than 90% of monocytes and neutrophils from healthy subjects, while the number of positive cells was decreased (60%) using the 109# MoAb. However, either 68# and 100# MoAbs recognised only a low number of blood monocytes and neutrophils (8-20%), while lymphocytes were unreactive with all the five UPA-R MoAbs. ALL cells were found to be CD87 negative in all cases. Blasts from AML showed a heterogeneous pattern of expression for the UPA-R MoAbs, being the reactivity strictly dependent on the MoAb used, and, to a higher extent, on the degree and type of maturation of the blastic cells. The number of blasts recognising 3B10# and VIM5* MoAbs was significantly higher than that reacting with the remaining MoAbs irrespective of the FAB subtype. Since proteolytic enzymes, like UPA, play a key role in the dissolution of the extracellular matrix, and in facilitating the cell egress from the bone marrow, it is conceivable that the expression of the UPA-R could contribute to the invasive properties and, possibly, metastatic potential of leukemic cells.

Important considerations in using indicators to profile providers.

The demand is accelerating for information about the clinical performance of providers. In the more competitive and value-sensitive marketplace that is already developing, purchasers (consumers, employers, and insurers) of health care services will require more information to better assess the relative value of providers' (professional and hospital) services. The cornerstone of a wise, value-based strategy in selecting health care services is careful assessment of each provider's performance based on detailed, quantitative data in the form of clinical indicators. The use of indicators to profile the comparative performances of providers allows purchasers to compare as well as to influence provider performance.

Cytogenetics of hybrid acute leukemias.

Although the recognition of hybrid acute leukemia (HAL) is still controversial, several reports have described cytogenetic findings in these leukemias over the last 3 years. A distinct chromosomal profile appears to be associated with different immunologic subsets of HAL. The classical t(15;17), and inv(16) as well as abnormalities of the long arm of chromosome 5 and/or 7 are preferentially associated with acute myeloid leukemia (AML) with T-cell features; the t(8;21)(q22;q22), the Ph chromosome, and 11q23 rearrangements are more frequently found in AML with B-cell features; the Ph chromosome, t11q23 and 14q32 breaks without rearrangements of the immunoglobulin heavy chain gene may be associated with acute lymphoblastic leukemia (ALL) with myeloid markers. In addition, some chromosome aberrations may be encountered more frequently in acute leukemia with major phenotype deviations than in unselected cases of acute leukemia: namely the Ph chromosome, 11q23 rearrangements, and +13. These chromosome changes appear to be associated with a low complete remission (CR) rate. An association has been documented in some patients with ALL between the presence of the t(9;22) and a minor myeloid component consisting of 5-15% blast cells with myelomonocytic features, raising the possibility that a diagnosis of bilineal acute leukemia would be more appropriate in such cases. These patients appear to have a severe outcome with significantly lower CR rate than similar cases of Ph-positive ALL without a minor myeloid component.(ABSTRACT TRUNCATED AT 250 WORDS)

Twelve questions to ask about your outcomes monitoring system--Part II.

Commercial and customized outcomes monitoring systems designed to assess the results of care, whether clinical outcomes or resource use, are not all of equal value or equally appropriate for every use. In creating each system, its developers had to make critical decisions about such matters as definitions of outcomes for study, selection of patients, selection of data elements, methods and timing of data collection, and method of analysis and reporting. Each system represents a unique set of choices that were made. This series of two articles presents answers to 12 questions that will help users understand the basic workings of an outcomes monitoring system--to be able to distinguish good systems from the mediocre and the bad, and to make wise use of a system already in operation. In addition to the six questions presented in the April 1994 issue of Physician Executive, the following six questions are of critical importance in determining a system's value to you and your organization.

Twelve questions to ask about your outcomes monitoring system--Part I.

Outcomes monitoring is an integral part of any decision maker's information resources--the cornerstone of a provider's commitment to quality improvement or of a purchaser's strategy for seeking value. In their eagerness to obtain useful information about provider performance, purchasers and consumers naively may accept flawed evaluations and thereby create perverse incentives for providers that undermine the very qualities they wish to foster. Inaccurate or misleading information about provider performance will lead managers to reward the wrong behavior and so induce more of it. Inaccurate information also can discourage better providers whose performances are not recognized and can lead all providers to distrust and denounce clinical monitoring in general. When these things happen, the great value of outcomes monitoring systems as a tool for quality improvement is lost.

Transformation of the cryobehavior of rye protoplasts by modification of the plasma membrane lipid composition.

The freezing tolerance of protoplasts isolated from nonacclimated rye leaves (Secale cereale L. cv Puma) was significantly altered by using a pH-induced protoplastliposome fusion technique to modify the lipid composition of the plasma membrane. The increase in freezing tolerance was elicited by fusion with liposomes composed of either the total phospholipid fraction isolated from the plasma membrane of cold-acclimated leaves or single mono- or diunsaturated species of phosphatidylcholine (PtdCho). Of the PtdCho species tested, dilinoleoylphosphatidylcholine ([Lin(2)]PtdCho) and dilinolenoylphosphatidylcholine ([Lnn(2)]PtdCho) liposomes were the most effective; 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or dioleoylphosphatidylcholine liposomes were somewhat less effective; dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine liposomes had no effect. The increased freezing tolerance was the result of a transformation in the cryobehavior of the plasma membrane during freeze-induced osmotic contraction. In control nonacclimated protoplasts, osmotic contraction resulted in endocytotic vesiculation of the plasma membrane which was irreversible and resulted in lysis during osmotic expansion after melting of the suspending medium. In nonacclimated protoplasts fused with mono- or diunsaturated species of PtdCho, osmotic contraction resulted in the reversible formation of exocytotic extrusions of the plasma membrane-as normally occurs in protoplasts isolated from cold-acclimated leaves (acclimated protoplasts). In scanning electron micrographs, the morphology of the extrusions of nonacclimated protoplasts fused with [Lin(2)]PtdCho was virtually indistinguishable from that of the extrusions formed in acclimated protoplasts. These studies provide direct evidence that changes in the lipid composition of the plasma membrane are causally related to one facet of the cold-acclimation process.

Leaf anatomy and ultrastructure of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana.

Light-microscopic analysis of leaf clearings of the obligate Crassulacean-acid-metabolism (CAM) species Kalanchoe daigremontiana Hamet et Perr. has shown the existence of unusual and highly irregular venation patterns. Fifth-order veins exhibit a three-dimensional random orientation with respect to the mesophyll. Minor veins were often observed crossing over or under each other and over and under major veins in the mesophyll. Paraffin sections of mature leaves show tannin cells scattered throughout the mesophyll rather evenly spaced, and a distinct layer of tannin cells below the abaxial epidermis. Scanning electron microscopy showed that bundle-sheath cells are distinct from the surrounding mesophyll in veins of all orders. Transmission electron microscopy demonstrated developing sieve-tube elements in expanded leaves. Cytosolic vesicles produced by dictyosomes undergo a diurnal variation in number and were often observed in association with the chloroplasts. These vesicles are an interesting feature of cell ultrastructure of CAM cells and may serve a regulatory role in the diurnal malic-acid fluctuations in this species.