Cell kinetics of gliomas by serial stereotactic biopsy

The potential proliferative activity of glial tumors has been investigate by serial stereotactic biopsies and by "in vitro" 3H-thymidine incorporation procedure (labeling index, LI). The methodology of this combined approach and the preliminary results in 33 patients are reported. Cell kinetic data have been matched with the histological classification (W.H.O.).     

A new approach to DNA polymerase kinetics

Little is known of the detailed mechanisms of the polymerization reactions carried out by RNA and DNA polymerases. Besides technical reasons, there are mathematical difficulties not encountered in traditional enzymology. The product of the reaction after one polymerization step is also the substrate of the next step. A number of polymerases, isolated from various sources, have an exonuclease activity. The chain which is being synthesized may be either elongated or trimmed, and its growth has the character of a random walk. In this case, although the overall reaction scheme is more complex, the experiments are more informative, as every dNTP may be transformed into two distinct products: incorporated, or free dNMP.Having solved some of the mathematical difficulties of the random walk problem, we are able to propose a strategy for the study of the polymerization/excision kinetics. We measure the amount y(t) of nucleotide that is polymerized at time tand the amount x(t) of nucleoside monophosphate that has accumulated. When $\text{d}\text{y}\text{d}\text{x}$ is plotted against the concentration of dNTP, a curve is obtained with a characteristic shape, a straight line in a large number of cases. From there, kinetic constants can be estimated.The analysis is made in terms of four possible kinetic schemes. In the most elementary model there are only two rate constants, one for incorporation and one for excision. This model is a limiting case of all other models. The frayed-unfrayed model of Brutlag & Kornberg (1972), Hopfield's kinetic proofreading scheme (Hopfield, 1974), and the delayed-escape scheme (Ninio, 1975) are examined in detail, and we show how the kinetic experiments may in principle distinguish between the schemes. Our approach is illustrated with three experiments in which Escherichia coli DNA polymerase I acts on poly(dC), and poly(dT) · oligo(dA)₁₀.     

Cell proliferation kinetics in two human tumors grown in athymic nude mice.

The technique of labelled mitoses was used to investigate the cell proliferation kinetics in two human neoplasms, one a malignant melanoma and one a fibrosarcoma, transplanted to and grown serially in the athymic nude mutant (ANM) mouse. The experimental data obtained codrealted well with a theoretical percentage labelled mitoses curve based on the assumption that the time spent by a cell in each of the phases M, G1, S and G2 is described by four independent log-normal distributions. However, no unique second wave was defined by the experimental results. This means that only the deductions made about the duration of the G2 and S phases are reliable. The median duration of tma and the fibrosarcoma, respectively. By comparing these results with results published on cell cycle studies of transplantable animal tumors and human tumors in situ, it is concluded that the cell cycle parameters of a human tumor grown in the ANM mouse are close to those of the same tumor in the donor patient.     

Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.     

Cell adhesion during gastrulation : A new approach

In gastrulating sea urchin embryos, secondary mesenchyme cells at the tip of the advancing archenteron extend long narrow filopodia which probe the inner surface of the blastocoele wall, rejecting some surface contacts before adhering to other cells. After specific cell adhesions are made, contractions of the filopodia pull the leading tip of the archenteron to the opposite wall of the blastocoele with an accompanying elongation of the archenteron. A study was made of the biochemistry and morphology of the specific adhesions of filopodial extensions by injecting a variety of compounds into the blastocoele of living sea urchin gastrulae and observing their effects on filopodia and cell movements. A number of agents (proteases, lectins) caused specific filopodial detachment and subsequent archenteron regression. Fluorescein-conjugated lectins, including concanavalin A (conA) and wheat germ agglutinin (WGA) exhibited marked specificity of cell surface binding to specific regions (primary mesenchyme cells, blastocoele wall, etc.) of the embryo.     

Differential expression of E-cadherin gene in human neuroepithelial tumors

Cadherins are cell-to-cell adhesion molecules that play an important role in the establishment of adherent-type junctions by mediating calcium-dependent cellular interactions. The CDH1 gene encodes the transmembrane glycoprotein E-cadherin which is important in maintaining homophilic cell-cell adhesion in epithelial tissues. E-cadherin interacts with catenin proteins to maintain tissue architecture. Structural defects or loss of expression of E-cadherin have been reported as a common feature in several human cancer types. This study aimed to evaluate the expression of E-cadherin and their correlation with clinical features in microdissected brain tumor samples from 81 patients, divided into 62 astrocytic tumors grades I to IV and 19 medulloblastomas, and from 5 white matter non-neoplasic brain tissue samples. E-cadherin (CDH1) gene expression was analyzed by quantitative real-time polymerase chain reaction. Mann-Whitney, Kruskal-Wallis, Kaplan-Meir, and log-rank tests were performed for statistical analyses. We observed a decrease in expression among pathological grades of neuroepithelial tumors. Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than did neuroepithelial tumors. Expression of E-cadherin gene was higher in astrocytic than embryonal tumors (P = 0.0168). Low-grade malignancy astrocytomas (grades I-II) showed higher CDH1 expression than did high-grade malignancy astrocytomas (grades III-IV) and medulloblastomas (P < 0.0001). Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than grade I malignancy astrocytomas, considered as benign tumors (P = 0.0473). These results suggest that a decrease in E-cadherin gene expression level in high-grade neuroepithelial tumors may be a hallmark of malignancy in dedifferentiated tumors and that it may be possibly correlated with their progression and dissemination.     

A new approach for analysis of chemiluminescent kinetics of activated phagocytes in blood

Luminol chemiluminescence (LCL) is a simple, sensitive and time-saving tool to elucidate the oxidative activity of polymorphonuclear leukocytes (PMNL). In this study, a new approach for analysing the LCL kinetics, recorded from stimulated PMNL, and for a more accurate elucidation of their functional state, is proposed. This approach is based on the proposal that the LCL kinetics of stimulated PMNL is a result of time-probabilistic nature of the processes, leading to light emission. On this basis the LCL response was described by the parameters of a Poisson-type distribution. Relationships between these parameters and some factors influencing LCL kinetics (number of PMNL and erythrocytes, temperature, stirring) were investigated. Their development in time was different. It was suggested, that the two phases of the model LCL response were connected with extracellular and intracellular LCL. The terms and the advantages of this approach for analysis of phagocyte oxidative capacity are discussed.     

Cell kinetics of a chondrosarcoma.

The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G1, 10-5 hr; S, 9-5 hr; G2, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0-5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.     

Cell kinetics of irradiated experimental tumors: relationship between the proliferating and the nonproliferating pool.

The role of nonproliferating cells in tumor regeneration has been studied after subcurative doses of low L.E.T. irradiation. Radiation was applied in a single dose at three different levels; 0-47, 0-94 and 1-88 krad. Studies included estimation of the absolute number of cells per tumor, differential cell counts, and autoradiographic determination of kinetic variables, employing transplantable mouse mammary adenocarcinoma DBAH. Quantitative changes of morphologically defined proliferating and nonproliferating cell pools were followed at different time intervals after irradiation. Irradiation resulted in reduction of the number of cells in both pools, with apparent sparing of nonproliferating cells. The regenerative period started with a gradual increase in the number of cells in the proliferating pool, whereas the number of cells in the nonproliferating pool continued to fall in tumors irradiated with 0-94 and 1-88 krad. In the late phase of tumor regrowth, the increasing number of cells in the non proliferating pool corresponded to its replenishment by cell transition from the proliferating pool. In an effort to clarify whether cell transition from the nonproliferating to the proliferating pool may take place during the regrowth of radiation perturbed tumors, cell loss rates from both pools were estimated using experimental data. In addition to cell loses from the tumor as a whole, the 'net loss rate' of the nonproliferating pool reflects the rate of cell transition from the nonproliferating to the proliferating pool, minus the rate of transition in the opposite direction. A similar definition applies to cell loss rates from the proliferating pool. The results showed: (1) high losses in both pools, with excess losses in the proliferating during the early phase after irradiation; (2) in the early stage of regrowth after irradiation, the cell net loss rate f-or the nonproliferating pool increased, in contrast to the behavior of cell loss rate for the proliferating pool and the average cell loss rate for the tumor as a whole; (3) in the late stage of regrowth a decrease in net loss rate for the nonproliferating pool reflects the excess production of nonproliferating cells over control tumors. These results suggest that cell transition from the nonproliferating to the proliferating pool takes place at the beginning of tumor regrowth after subcurative single-dose irradiation.     

A new assay for tRNA aminoacylation kinetics.

An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3'-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive.     

A new approach in the kinetics of biological transport the potential of reversible inhibition studies

Kinetic equations are derived for reversible inhibition of both active and facilitated transport systems for seven common experimental arrangements. It is shown that the unique features of transport kinetics may be exploited to give new kinds of information. It is also shown that the familiar rules of enzyme kinetics, though often applied to transport, can be seriously misleading. The analysis leads to the following general conclusions: (1) A competitive mechanism frequently gives rise to non-competitive kinetics, depending on the experimental design, but a non-competitive mechanism never produces competitive kinetics. (2) Inhibition studies on exchange diffusion at equilibrium in non-active systems or in the final steady state in active systems are the only unambiguous kinetic tests to distinguish competitive from non-competitive mechanisms. (3) Substrate analogs that are bound to the carrier and transported are readily distinguished by inhibition kinetics from those not transported, even though both may rapidly enter the cell by another route. (4) Even in non-active systems competitive inhibitors commonly have far different affinities for the substrate sites on the two membranes faces: where sufficient non-polarity allows their penetration into the cell, inhibition kinetics readily establish such sidedness in their action. (5) Inhibition kinetics of the mixed competitive and non-competitive type result from moderately asymmetrical binding of inhibitor at the substrate site. (6) Asymmetry is a necessary feature of active transport; hence studies of inhibition kinetics should provide important insights into its mechanism.     

A new head clamp for stereotactic and intracranial procedures. Technical note

The authors have devised a clamp to fix the head to the surgical table during stereotactic procedures for placement of depth electrodes. The main advantages of this device derive from the rigid immobilization of the head. The clamp facilitates fixation of the stereotactic frame to the head, percutaneous twist drill trephination of any site of the skull, X-ray centering with a laser beam, mobilization of contrast material during ventriculography or pneumoencephalography, and placement of epidural screws. This head clamp can also be used during craniotomies for fixation of the head and support for brain retractors.     

Paratesticular adenomatoid tumors. The cytologic presentation in fine needle aspiration biopsies

Adenomatoid tumors are the most common tumors of male paratesticular tissues (epididymis, tunica or spermatic cord) and have also been described in females (uterus, fallopian tube, ovary and paraovarian tissues); fine needle aspiration (FNA) of masses in these locations is increasingly utilized as an alternative to surgical exploration in order to establish a tissue diagnosis. This paper describes the FNA cytodiagnosis of seven cases of paratesticular adenomatoid tumors. The main cytologic criteria included epithelioid sheets and multilayered clusters of monotonous cells with round or ovoid, eccentric nuclei containing small, central nucleoli. Paranuclear clearing with a pink coloration (Giemsa stain) or a clear vacuolelike area (Papanicolaou stain) and abundant cellularity with a background of naked nuclei and stromal fragments were noted. The clinical presentation and clinicohistologic follow-up of these seven cases is also described in detail. A discussion of the differential diagnosis and the expected FNA findings is provided.