Cytogenetic evidence for premeiotic transformation of human testicular cancers.

To determine the point at which transformation of the germ cell occurs during meiosis in nonseminomatous testicular cancer, the sex chromosome compositions of 15 cell lines derived from primary tumors or metastases of 12 patients with testicular cancer were analyzed by trypsin G-banding analysis and Y-body staining. The simultaneous existence of both X- and Y-chromosomes in a single cell has been confirmed in 14 cell lines. This suggests that transformation of the cell occurs before the first meiotic division because it is known that segregation of X- and Y-chromosomes occurs during the first meiotic division. An incidental finding was the presence of Barr bodies in some cell lines containing more than one X-chromosome, which is consistent with the known primitive nature of testicular cancer and its ability to differentiate independently from the male host.

Involvement of band 3p14 in t(3;8) hereditary renal carcinoma.

High resolution prometaphase G-banding analysis was applied to three translocation carriers from the t(3;8) hereditary renal cell carcinoma family. It was clearly illustrated that the chromosomal rearrangement is reciprocal with breakpoints occurring at the subbands 3p14.2 (instead of 3p21) and 8q24.1.

Clinical efficacy and tolerance of an extract of green-lipped mussel (Perna canaliculus) in dogs presumptively diagnosed with degenerative joint disease.

AIM: To evaluate the efficacy and tolerance of an extract of green-lipped mussel (GLME) in the management of mild-to-moderate degenerative joint disease (DJD) in dogs. METHODS: Eighty-one dogs presumptively diagnosed with DJD were treated orally daily with either GLME or a placebo for 56 days, in a double-blind, placebo-controlled study. In an uncontrolled open-label extension to the study, all dogs were treated with GLME for an additional 56 days (from Days 57-112). Clinical signs were subjectively scored by the owners, and findings of detailed musculoskeletal examinations were scored by one veterinarian. Efficacy was assessed from a qualitative comparison of the proportion of dogs with improved clinical signs, and a quantitative comparison of the scores of the musculoskeletal examinations, between groups. Haematological and biochemical analyses and reports by owners of possible adverse drug reactions were used to screen for evidence of toxicity. RESULTS: There was close agreement between assessments by the veterinarian and owners. The clinical signs of DJD in both GLME-treated and placebo groups improved significantly over baseline by Day 28; this improvement continued over the entire course of the study. There were no significant differences between groups on Day 28. On Day 56, a higher proportion of dogs in the GLME-treated group had improved clinical signs (p=0.018), and GLME-treated dogs had marginally better (p=0.053) musculoskeletal scores than dogs in the placebo group. The differences between the groups were no longer apparent by Day 112, by which time the former placebo group had been receiving GLME for 56 days in the open-label phase of the study. The proportion of dogs in the former placebo group that had improved by Day 112 (29/32; 91%) was significantly greater (p=0.012) than the proportion improved at Day 56 (15/37; 41%). No signs of toxicity were apparent. CONCLUSIONS AND CLINICAL RELEVANCE: GLME had a beneficial effect on the clinical signs of dogs presumptively diagnosed with mild-to-moderate DJD. Long-term therapy may be required before improvement is apparent.

Cl- accumulation does not account for the depolarizing phase of the synaptic GABA response in hippocampal pyramidal cells.

It has been proposed that the depolarizing phase of the biphasic synaptic GABA response could be mediated by HCO3- passing through GABA(A) channels after dissipation of the transmembrane Cl- gradient due to intracellular Cl- accumulation. To test this hypothesis, giant GABA-mediated postsynaptic currents (GPSCs) were recorded from pyramidal cells in slices of adult guinea pig hippocampus in the presence of 4-aminopyridine. GPSCs consisted of an early outward current (GABA(A) component) followed by a late inward current (GABA(D) component). Spontaneous outward inhibitory postsynaptic currents (IPSCs) occurred during the GABA(D) component of the GPSC. GPSCs that were evoked 1-12 s after the preceding GPSC (short interval, siGPSCs) showed no GABA(D) component even though in many cells the amplitude of the siGPSC was greater than the amplitude of the GABA(A) component of the preceding spontaneous GPSC. In addition, the siGPSC evoked during the GABA(D) component of a spontaneous GPSC was an outward current. To test whether the siGPSC lacked a GABA(D) component because it was generated predominantly at the soma, where less of an increase in [Cl-](i) would occur, picrotoxin was applied to the soma of the pyramidal cell. To the contrary, this focal application of picrotoxin caused less of a reduction in the amplitude of the siGPSC than in the amplitude of the GABA(A) component of the GPSC. Furthermore when a GPSC and siGPSC were evoked 10 s apart using identical stimuli, the area under the outward current curve was sometimes greater for the siGPSC than for the GPSC, and yet the siGPSC had no inward component. This result indicates that even when the location of Cl- entry was the same, more Cl- could enter the cell during the siGPSC than during the outward component of the GPSC and yet not lead to an inward current. In addition, when the second of two identical stimuli was applied during the inward GABA(D) component of the first evoked GPSC, the GABA(A) response it generated was always outward, demonstrating that the equilibrium potential for GABA(A) responses did not become more positive than the holding potential during a GPSC. Finally, evoking GPSCs at a hyperpolarized potential revealed that the siGPSC actually lacked a GABA(D) conductance. These results disprove the Cl- accumulation hypothesis of the synaptic depolarizing GABA response and suggest the possibility that a separate channel type may mediate the GABA(D) component of the GPSC.

Ionic basis of the postsynaptic depolarizing GABA response in hippocampal pyramidal cells.

1. Whole cell voltage-clamp recording with recording pipette solutions of differing ionic composition was used to determine the ionic basis of the depolarizing gamma-aminobutyric acid (GABA) response. In the presence of 4-aminopyridine and excitatory amino acid receptor blockers, giant GABA-mediated postsynaptic currents (GPSCs) were recorded from CA3 pyramidal neurons in hippocampal slices from adult guinea pigs. With the GABAB component blocked, the GPSC was composed of an initial outward current (GABAA component) that peaked at 115 ms followed by a late inward current (GABAD component) that peaked at 400-600 ms. 2. Reduction of the intracellular concentration of potassium ([K+]i)resulted in no significant change in the reversal potential of the GABAD component of the GPSC, indicating that it is not a nonspecific cation current. 3. The HCO3- permeability of the channel mediating the GABAD response was assessed by using recording pipette solutions containing three different concentrations of bicarbonate ([HCO3-], 19, 49, and 102 mM). The reversal potential of the GABAD response shifted in the depolarizing direction as the HCO3- equilibrium potential was shifted in the depolarizing direction, indicating that the channel mediating the GABAD response is permeable to HCO3-. The reversal potential of the GABAD response was more sensitive to changes in recording pipette [HCO3-] than the reversal potential of the GABAA response, indicating that the GABAD response is carried by HCO3- to a greater extent than the GABAA response. 4. The outward current-inward current sequence of the biphasic GPSC was reversed to an inward current-outward current sequence by using a high [Cl-]/low [HCO3-] recording pipette solution (40 mM Cl-/6 mM HCO3-), indicating that the GABAA component is more sensitive to changes in [Cl-]i, and the GABAD component is more sensitive to changes in [HCO3-]i. 5. These data indicate that the GABAD component of the GPSC is predominantly carried by HCO3-. While this result supports the recently propsed chloride accumulation model, the model in its present form cannot explain the inward current-outward current polarity sequence of the GPSC recorded with the high [Cl-]/low [HCO3-] intracellular solution. The data obtained using that solution reveal the need for a more expansive chloride accumulation/ depletion model or for a model utilizing two distinct ionotropic GABA channels with different anion permeability ratios to account for the biphasic nature of the GPSC.

The depolarizing GABA response.

In some situations the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) elicits a hyperpolarizing response (H response) followed by a depolarizing response (D response) on cortical neurons. It has recently been established that the D response on hippocampal pyramidal cells is largely carried by bicarbonate ions (HCO3-). However, there is still controversy over whether the hyperpolarizing and depolarizing responses to GABA are mediated by the same receptor channel. A relatively new single receptor channel model proposes that the D response appears because chloride (Cl-) accumulates as a results of the Cl(-)-HCO3- conductance mediating the GABA response, dissipating the Cl- driving force and unmasking a net inward HCO3- current. This chloride accumulation model has gained favor because it provides an explanation for why higher concentrations of GABA are required to elicit the D response and why the D response generally follows an H response. On the other hand, there are some data that are best explained if there are two separate types of receptor channels mediating the H and D responses. This paper presents evidence for and against both the chloride accumulation hypothesis and the two different receptor channels hypothesis in the hope that investigators will recognize that this key problem concerning the generation of the D response remains unsolved.

Intracellular QX-314 blocks the hyperpolarization-activated inward current Iq in hippocampal CA1 pyramidal cells.

1. Whole cell voltage-clamp recordings (access resistance < or = 12 M omega) from CA1 pyramidal cells in the guinea pig hippocampal slice revealed a hyperpolarization-activated inward current with an inward tail upon repolarization. The current activation range extended from approximately -50 mV to -130 mV, with half-activation at -86 mV. This current was identified as the q current (Iq). 2. Intracellular QX-314 (5 or 10 mM), a quaternary derivative of lidocaine, blocked Iq completely throughout its activation range. 3. There is a growing realization that Iq may be responsible for the pacemaker depolarization in cells that display rhythmic calcium spikes. Because QX-314 blocks Iq completely, it could be used to test whether Iq is essential to this oscillatory activity.

Zn2+ blocks the NMDA- and Ca2+ -triggered postexposure current ipe in hippocampal pyramidal cells.

Whole cell voltage-clamp recordings from acutely isolated hippocampal CA1 pyramidal cells from adult guinea pigs were used to evaluate divalent cations as possible blockers of the postexposure current (Ipe). Ipe is a cation current that is triggered by the rise in intracellular Ca2+ concentration that occurs after the application of a toxic level of N-methyl-D-aspartate (NMDA). Once triggered, Ipe continues to grow until death of the neuron occurs. Ipe may be a critical link between transient NMDA exposure and cell death. Ipe was blocked by micromolar concentrations of Zn2+. The Zn2+ effect had an IC50 of 64 microM and saturated at 500 microM. Prolonged Zn2+ block of Ipe revealed that the maintenance of a steady Ipe is not dependent on Ipe-mediated Ca2+ influx but that the continuous growth in Ipe is dependent on Ipe-mediated Ca2+ influx. The availability of an effective blocker of Ipe should facilitate the investigation of the intracellular activation pathway of Ipe and the role of Ipe in neuronal death.

Secondary activation of a cation conductance is responsible for NMDA toxicity in acutely isolated hippocampal neurons.

One of the key questions concerning glutamate toxicity is how a transient NMDA exposure can lead to a delayed death of neurons. To address this issue, we performed whole-cell recording on acutely isolated hippocampal CA1 neurons to monitor the membrane response after NMDA exposure. Transient NMDA exposure (100 microM, 10 min) induced an inward current (postexposure current; Ipe) which was associated with a Ca2+- and Na+-permeable cation conductance. Ipe continuously increased (in the absence of NMDA) until death of the neuron occurred. Application of NMDA in the absence of extracellular calcium failed to trigger Ipe and neuronal death. Postexposure suppression of Ipe protected against NMDA toxicity. These results indicate that a cation current, which is induced by an increase in intracellular calcium concentration ([Ca2+]i) and is itself partly carried by Ca2+, links the initial NMDA exposure to neuronal death.

Evaluation of colostrum and whey-derived gamma globulins for prevention of cryptosporidiosis in artificially infected neonatal calves.

Thirty colostrum-deprived Friesian male calves were used to study the effect of colostrum and whey-derived gammaglobulins on the clinical events and oocyst-excretion pattern following artificial infection with a fresh, field-sourced strain of Cryptosporidium parvum. The calves were raised naive and free from contact with C. parvum from birth until infection at either 10 or 17 days of age. The two age groups comprised three sub-groups that were each given a single treatment of colostrum, whey-derived gammaglobulins or whole milk (controls) at 5 hours after birth. Blood samples taken both before and 48 hours after this dosing showed the mean serum gamma globulin concentrations changed from almost zero in all calves to 462,279 and 29 mg/dl for the colostrum, whey-derived gammaglobulin and whole milk sub-groups respectively. The results showed that a majority of calves shed Cryptosporidium oocysts within 7 days of oral infection but that no diarrhoea or other clinical signs were associated with this. However, when the level of C. parvum faecal shedding was graded, the results showed a trend towards a higher level of oocyst shedding and over a longer period of time in the control calves deprived of any passive immunity than in the two groups given either colostrum or whey-derived gammaglobulins.

Relationship between blood selenium concentration or glutathione peroxidase activity, and milk selenium concentrations in New Zealand dairy cows.

AIM: To determine the relationships between blood selenium (Se) concentrations or glutathione peroxidase activity (GSH-Px), and milk Se concentrations in dairy cows. METHODS: Seventy-two Friesian dairy cows were either untreated or injected with 0.5, 1.0 or 2.0 mg Se/kg liveweight as barium selenate (BaSeO4) formulations, resulting in 6 groups of animals with mean blood Se concentrations that varied from 212 to 2272 nmol/l. Milk samples were collected on Days 104 and 188, and blood samples were collected prior to treatment and on Days 41, 76, 104, 188, 244, and 292 after Se injection. RESULTS: Significant quadratic relationships between blood Se and milk Se concentrations, as well as blood GSH-Px activity and milk Se concentrations, were evident at Days 104 and 188. Using combined data, these were represented by the equations: milk Se = 27.3 + 0.073 blood Se -0.00001 (blood Se)2; R2=0.79, p<0.005, and; milk Se = 34.8 + 4.99 GSH-Px -0.068 (GSHPx)2; R2=0.79, p<0.005. CONCLUSIONS: The Se status of dairy cows can be assessed from milk Se concentrations. CLINICAL SIGNIFICANCE: Bulk-tank milk Se concentrations could be evaluated as a method to assess the Se status of dairy herds.