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Biomedical subjects

M Malecki

Publications and source records attributed to M Malecki.

16 recordsLinked to original sources
PubMed2005

Angiogenic and antiangiogenic gene therapy.

Gene therapy is thought to be a promising method for the treatment of various diseases. One gene therapy strategy involves the manipulations on a process of formation of new vessels, commonly defined as angiogenesis. Angiogenic and antiangiogenic gene therapy is a new therapeutic approach to the treatment of cardiovascular and cancer patients, respectively. So far, preclinical and clinical studies are successfully focused mainly on the treatment of coronary artery and peripheral artery diseases. Plasmid vectors are often used in preparations in angiogenic gene therapy trials. The naked plasmid DNA effectively transfects the skeletal muscles or heart and successfully expresses angiogenic genes that are the result of new vessel formation and the improvement of the clinical state of patients. The clinical preliminary data, although very encouraging, need to be well discussed and further study surely continued. It is really possible that further development of molecular biology methods and advances in gene delivery systems will cause therapeutic angiogenesis as well as antiangiogenic methods to become a supplemental or alternative option to the conventional methods of treatment of angiogenic diseases.

Animals↗
PubMed2001

In vitro and in vivo study of the expression vector encoding vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine with potential therapeutic applications in human diseases. It is a mitogen primarily for endothelial cells. The transfer of the cDNA encoding VEGF to ischemic tissues, which cannot be revascularized otherwise, represents a novel and promising approach to the treatment of vascular disorders. In this work the VEGF165 cDNA was cloned into the expression vector pSecTag2B. The activity of the construct was studied in cell culture as well as in vivo. Western blotting study showed that the cells transfected with the vector secreted significantly higher amounts of VEGF to the culture medium than the non-transfected cells. In vivo study revealed an increased number of new vessels in animals injected with vector encoding VEGF as compared with empty plasmid. Also, tumor cells transfected with the VEGF plasmid exhibited extensive vascularization.

Animals↗
PubMed1999

Amino acid variants of the vitamin D-binding protein and risk of diabetes in white Americans of European origin.

BACKGROUND: Genetic variants of vitamin D-binding protein (DBP) have been reported to be associated, not only with diabetes, but also with prediabetic traits, in several populations. There are two known polymorphisms in exon 11 of the DBP gene that result in amino acid variants: at codons 416 GAT-->GAG (Asp-->Glu) and 420 ACG-->AAG (Thr-->Lys). OBJECTIVE: To examine the association of these polymorphisms with diabetes in white Americans of European origin. METHODS: We studied unrelated individuals: 181 with type 1 diabetes, 215 with type 2 diabetes, and 163 healthy controls. Exon 11 was amplified using polymerase chain reaction and the two alleles were determined by digestion with specific endonucleases: HaeIII and StyI, respectively. RESULTS: At codon 416, Asp/Glu allele frequencies were 45%/55% in patients with type 1 diabetes, 43%/57% in patients with type 2 diabetes, and 46%/54% in controls (chi(2)=0.69, 2 d.f., P<0.71). At codon 420, corresponding Lys/Thr frequencies were 27%/73%, 30%/70%, and 30%/70% (chi(2)=1.25, 2 d.f., P=0.53). Distributions of genotypes at both loci, and the haplotypes defined by the two loci, were also very similar in all groups. CONCLUSION: DNA polymorphisms in the DBP gene are not associated with diabetes in white Americans of European origin.

Adult↗
PubMed1999

Phenotypic characteristics of early-onset autosomal-dominant type 2 diabetes unlinked to known maturity-onset diabetes of the young (MODY) genes.

OBJECTIVE: To investigate whether there are forms of early-onset autosomal-dominant type 2 diabetes that are distinct from typical maturity-onset diabetes of the young (MODY) and to characterize their phenotypic characteristics. RESEARCH DESIGN AND METHODS: The study included 220 affected subjects from 29 families in which early-onset type 2 diabetes occurred in multiple generations and was not linked to known MODY genes (MODY gene-negative families). All individuals underwent an oral glucose tolerance test and other clinical measurements aimed at investigating the underlying metabolic defect and the presence of diabetic complications. For comparison, 79 affected carriers of MODY3 (hepatocyte nuclear factor [HNF]-1 alpha) mutations were similarly examined. RESULTS: Subjects from MODY gene-negative pedigrees were diagnosed with diabetes at an older age (36 +/- 17 vs. 21 +/- 10 years, P = 0.0001) and were more frequently obese (52 vs. 18%, P = 0.0001) than MODY3 individuals. MODY gene-negative patients who were insulin treated required more exogenous insulin than did MODY3 subjects (0.7 +/- 0.4 vs. 0.45 +/- 0.2 U.kg-1.day-1, P = 0.04), despite similar C-peptide levels. Among subjects not treated with insulin, MODY gene-negative subjects had significantly higher serum insulin levels, both fasting (16.5 +/- 15 vs. 6.5 +/- 5 microU/ml, P = 0.027) and 2 h after a glucose load (53 +/- 44 vs. 11 +/- 10, P = 0.002). They also had higher serum triglycerides (P = 0.02), higher cholesterol levels (P = 0.02), more hypertension (P = 0.0001), and more nephropathy (P = 0.001). Differences persisted when families were matched for age at diagnosis. CONCLUSIONS: Our findings indicate the existence of forms of early-onset autosomal-dominant type 2 diabetes that are distinct from MODY and are frequently characterized by insulin resistance, similar to later-onset type 2 diabetes. Because of the Mendelian pattern of inheritance, the goal of identifying the genes involved in these forms of diabetes appears to be particularly feasible.

Adult↗
PubMed1997

New susceptibility locus for NIDDM is localized to human chromosome 20q.

To test the hypothesis that a gene (or genes) in the "MODY1 region" of the long arm of chromosome 20 contributes to the development of NIDDM, we conducted linkage studies in 29 extended Caucasian families in which many members were affected with NIDDM. A total of 498 individuals, including 159 NIDDM patients with an average age at diagnosis of 47 years, were genotyped for eight highly polymorphic microsatellite markers spanning a 31-cM region on chromosome 20q12-13.1. Using affected sib-pair analysis, we obtained evidence suggesting linkage between NIDDM and markers D20S119, D20S178, and D20S197 (allele sharing identical-by-descent [IBD], 0.56 for all three; P = 0.005, P = 0.009, and P = 0.004, respectively). Multipoint nonparametric linkage (NPL) analysis also showed evidence for linkage of NIDDM with the same three markers. The evidence for linkage was much stronger (allele sharing IBD by affected sibpairs, 0.64 [P < 0.0001]; maximum NPL score, 3.3 [P = 0.009]) in the 14 families whose average age at diagnosis of NIDDM was above the median (47 years) for all families. In these 14 families, one particular allele of the microsatellite D20S197 was transmitted from heterozygous parents to NIDDM offspring more frequently than expected (P < 0.01). This indicates that the marker allele and the disease allele are in linkage disequilibrium, implying that they are in close proximity. Consequently, the recently identified MODY1 gene (hepatocyte nuclear factor 4) is an unlikely candidate gene for NIDDM in our families, since it is located about 8 cM centromeric of D20S197. In conclusion, we have identified a new region on chromosome 20q that contains one or more NIDDM genes distinct from the recently identified MODY1 gene.

Adult↗
PubMed1996

Preparation of plasmid DNA in transfection complexes for fluorescence and electron spectroscopic imaging.

The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. Ultra-structural distribution of the transfected DNA was imaged with energy filtering transmission electron microscopy. In the second method, the unmodified transfected DNA was detected in cryo-immobilized cells by in situ polymerase chain reaction and in situ hybridization. For laser scanning fluorescence microscopy probes were labeled with tetramethylrhodamine. For ultrastructural analysis by electron spectroscopic imaging, probes containing incorporated digoxigenin were labeled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the presence of the nuclear localization signal in the transfection complex resulted in rapid nuclear import of the transfected DNA.

Cryopreservation↗
PubMed1993

Scanning electron microscopy of muscle myofibrils after high pressure freezing and freeze-substitution-staining.

A novel approach to study the three dimensional ultrastructure of organelles and cells by means of scanning electron microscopy is described. Muscle myofibrils have been used in the development of the techniques since their structure is well characterized using conventional electron microscopic methods. Myofibrils in rigor buffer (with no cryo-protectants or pressure sealants) were frozen at high pressure (2300 bar) within specially designed chambers. The frozen specimens were then freeze-substituted-stained with methanol containing tungsten and iron salts and finally critical point dried. These methods allowed scanning electron microscopic observations of the organization of individual filaments within whole myofibrils over several sarcomeres. Images obtained showed excellent structural preservation with three dimensional information which is not available with other electron microscopic techniques. Success in these approaches was ascribed to (a) rapid and uniform freezing at high pressure without ice segregation patterns, (b) uniform electro-conductivity of the specimen closely attached to the polished carbon piston/carrier, and (c) good electron emission (secondary and back-scattered) from the metal incorporated into the myofibril structure without additional coating.

Animals↗
PubMed1993

Scanning electron microscopy of high-pressure-frozen sea urchin embryos.

High-pressure-freezing permits direct cryo-fixation of sea urchin embryos having a defined developmental state without the formation of large ice crystals. We have investigated preparation protocols for observing high-pressure-frozen and freeze-fractured samples in the scanning electron microscope. High-pressure-freezing was superior to other freezing protocols, because the whole bulk sample was reasonably well frozen and the overall three-dimensional shape of the embryos was well preserved. The samples were either dehydrated by freeze-substitution and critical-point-drying, or imaged in the partially hydrated state, using a cold stage in the SEM. During freeze-substitution the samples were stabilized by fixatives. The disadvantage of this method was that shrinking and extraction effects, caused by the removal of the water, could not be avoided. These disadvantages were avoided when the sample was imaged in the frozen-hydrated state using a cold-stage in the SEM. This would be the method of choice for morphometric studies. Frozen-hydrated samples, however, were very beam sensitive and many structures remained covered by the ice and were not visible. Frozen-hydrated samples were partially freeze-dried to make visible additional structures that had been covered by ice. However, this method also caused drying artifacts when too much water was removed.

Animals↗
PubMed1991

Early results using high-resolution, low-voltage, low-temperature SEM.

Recent advances in the design of the scanning electron microscope (SEM) column, such as the coupling of a field-emission gun to a low-aberration immersion lens and the availability of a high-stability cryo-transfer stage, make low-temperature, low-voltage SEM (LTLVSEM) possible at very high resolution. We have used this combination to obtain results with uncoated biological specimens. The trichocyst from a Paramecium was used as a test specimen to observe the shrinkage of this structure as the temperature is raised from 170 K to room temperature following freeze-drying. High-magnification stereo images were obtained of trichocysts that had been prepared by freezing, freeze-substitution and critical-point drying and which were subsequently viewed by LTLVSEM to reduce beam damage and contamination.

Animals↗
PubMed1991

High voltage electron microscopy and low voltage scanning electron microscopy of human neoplastic cell culture.

Improved procedures were developed to correlate cell culture data with the images provided by advanced ultrastructural technologies. These procedures were compatible with the two main types of cellular behavior: adherent, spreading (melanomas, rhabdomyosarcomas) and non-adherent in suspension (leukemias). The ultrastructure and function of spreading neoplastic cells primarily depend on surface properties of the attaching substrates. Therefore, the films used for cultured cell whole-mount ultrastructural analysis must have adherence features identical to those of standard cell culture vessels. Improved procedures were developed to produce the polystyrene films of required qualities. These films allowed processing of cells for electron microscopy including chemical fixation, cryo-immobilization, and immunolabelling. Furthermore, these polystyrene films permitted observations of the same cell in the high voltage electron microscope to reveal the internal organization and in the low voltage scanning electron microscope to reveal the surface topography. Neoplastic cells in suspension may dramatically change their ultrastructure as a result of interactions with substrates or other cells. Therefore, immobilization of cellular processes must occur rapidly while cells remain in suspension. These processes were cryo-immobilized by high pressure freezing through the use of the newly designed specimen carrier. Procedures allowing high yield attachment of cryo-fixed neoplastic cells to amino-propyl-derived glass carriers enabled observations of cell surface topography. Furthermore, freeze-substitution and drying of freeze-fractured cells revealed their three-dimensional internal organization in the low voltage scanning electron microscope.

Cryopreservation↗
PubMed1987

Hospitals to receive help in organ procurement.

Healthcare professionals, including physicians, nurses, and clergy, must know how to initiate the process of organ and tissue donation to fulfill a patient's or family's wishes and how to ensure that the organs and tissues recovered by transplant teams are medically acceptable. After Oct. 1, 1987, federal law requires that hospitals have an organ donation policy and a mechanism in place for contacting an organ procurement agency (OPA). Non-compliance could affect Medicare and Medicaid reimbursement. An OPA is responsible for educating its referral hospitals about donor criteria, the national donor-recipient matching system, and how to discuss donation with grieving families. As part of this process, healthcare professionals can be involved in formal educational sessions that address the following questions: What is meant by "organ" and "tissue"? Who can be an organ or tissue donor? Do healthcare professionals need to be able to recognize a potential donor and handle everything on their own? What procedures are followed in initiating the discussion about donation with the family? How should one offer the option of organ and tissue donation to a bereaved family? What are some common family concerns and questions? What does donor maintenance entail? What happens in the operating room? Is there any follow-up after transplantation?

Bereavement↗
PubMed1976

Duodenal contraction wave patterns in patients with and without ulcer.

To determine whether differences in frequency of specific wave sequences and types existed in the four parts of the duodenum in ulcer subjects and controls an analysis of contraction waves in 14 controls and 10 duodenal ulcer patients of similar age was done by a statistical analysis of differences between the two groups. A water-perfused tube system was used. Differences in motor activity, namely, decreased frequency of waves in the proximal duodenum and increased frequency of waves in the distal duodenum in ulcer subjects as compared to controls was demonstrated. This involved a statistically significant decrease in numbers of mixing (or basic rhythm) waves in the proximal duodenum. Increased numbers of isolated waves were found in ulcer patients in the distal duodenum. The predominant types of contraction waves in both ulcer and control subjects were isolated and simultaneous waves, with peristaltic waves were less common in the fasting state. These findings suggest that differences exist in the types of contraction waves in duodenal ulcer subjects compared to controls.

Duodenal Ulcer↗
PubMedDate not supplied

High-resolution field emission scanning electron microscope imaging of internal cell structures after Epon extraction from sections: a new approach to correlative ultrastructural and immunocytochemical studies.

The availability of high-resolution field emission scanning electron microscopes (FESEM) and the recent development of a less destructive method for extracting Epon from sections motivated us to investigate these techniques for the study of internal cell structures. We chose the nuclear pore complex (NPC) and insect striated muscle as test objects. Chemically fixed or rapidly cryoimmobilized samples were embedded in Epon 812. The Epon was extracted from 200- to 300-nm-thick sections with a modified potassium methoxide-crown ether complex. The samples were viewed with high-resolution FESEM at low voltages. In tangential sections of isolated nuclear envelopes from Xenopus oocytes the cytoplasmic and intranuclear components ("fishtraps") of NPCs appeared identical to what has been described from whole mounts. In cross sections, fishtraps are seen in side view, which is possible only with this technique. In longitudinal and cross sections of insect flight muscle the classical arrangement of myofilaments and cross-bridges is well preserved. This method now makes it possible to image internal cell structures from any desired angle by high-resolution FESEM. Immunolabeling studies on the rabbit psoas muscle demonstrated that antigenicity of alpha-actinin was retained in Epon-extracted sections. Immunogold labeling with antibodies against alpha-actinin conjugated to 3-nm gold beads was intense, highly specific, and restricted to the Z lines. This method can overcome the penetration problem of immunogold labeling, since any cell component can be positioned at the surface of the section. Obviously this approach can become a powerful new tool for many areas of structural cell biology.

Actinin↗
PubMedDate not supplied

Signal transduction in adherent and non-adherent human cell lines after fibronectin stimulation.

It is shown that adherent and non-adherent human ovarian carcinoma cells (OVP 10) secrete MMPs and their production was stimulated by fibronectin as documented by gelatinise zymography. These cells also presented an increase of ERK phosphorylating activity following fibronectin stimulation, regardless of their adhesion. Contrary to OVP 10 cells, the human urothelial cells (HCV-29) are more anchorage-dependent. They only secreted the MMPs under adherent conditions and they revealed a lower level of basal and fibronectin stimulated ERK phosphorylation activity. In addition, non-adhering HCV-29 cells showed post translational down-regulation of focal adhesion kinase.

Blotting, Western↗